OCT-3 polypeptides

ABSTRACT

The invention relates to OCT-3 polypeptides, nucleic acid molecules encoding OCT-3, and uses thereof. OCT-3 is a protein that is expressed in the plasma membrane of biological cells, across which it regulates the transport of organic molecules.

RELATED APPLICATION INFORMATION

This application is a divisional of U.S. application Ser. No.08/964,127, filed Nov. 6, 1997 now abandoned.

BACKGROUND OF THE INVENTION

The field of the invention is cellular transporter molecules.

In the course of performing their normal physiological functions, manytypes of cells, including bacterial cells and those in specializedmammalian tissues such as the liver and kidney, transport a variety oforganic molecules across their cell membranes. For example, cells in theproximal tubule of the kidney transport glucose, amino acids, and uricacid across their membranes, and work to eliminate various drugs andtoxic substances from the body. All of these molecules are transportedacross the cell membranes by specialized cellular transporters.

Recently, genes encoding several putative transporters have beenidentified. These molecules include OCT-1 (organic cation transporter;Grundemann et al., Nature 372:549-552, 1994), OCT-2 (Okuda et al.,Biochem. Biophys. Res. Comm. 224:500-507, 1996), NLT (novelliver-specific transporter; Simonson et al., J. Cell Sci. 107:1067-1072,1994), and NKT (novel kidney-specific transporter; Lopez-Nieto et al.,J. Biol. Chem. 272:6471-6478, 1997). While the sequences of thesetransporters are not highly conserved (at the amino acid level, OCT-1and NLT are only 30% and 35% identical to NKT, respectively), they doexhibit similar transmembrane (TM) domain hydropathy profiles.

SUMMARY OF THE INVENTION

The invention described herein relates to the discovery andcharacterization of oct-3, a gene encoding a protein that transportsmolecules across the plasma membranes of biological cells. OCT-3 ishighly expressed in the neuronal cells of the brain. Accordingly,altering the expression or activity of OCT-3 (e.g., with smallmolecules, antisense molecules, or neutralizing antibodies) can alterthe concentration of molecules (such as neurotransmitters) that-arepresent within the cell or in the extracellular spaces around the cell(i.e., on either side of the plasma membrane). Altering theconcentrations of these molecules in patients afflicted with certainconditions, including neurodegenerative diseases, behavioral disorders,and eating or sleep disorders, can provide relief from the symptomsassociated with these conditions.

More specifically, the invention features an isolated nucleic acidmolecule (i.e., a nucleic acid molecule that is separated from the 5′and 3′ coding sequences with which it is immediately contiguous in thenaturally occurring genome of an organism) that encodes an OCT-3polypeptide. As used herein, an OCT-3 polypeptide is a polypeptide that:(1) is expressed in the plasma membrane of a biological cell (e.g., acell in the kidney, liver, or brain), (2) contains TM domains, and (3)when functioning normally, transports organic molecules across theplasma membrane. Preferably, the OCT-3 polypeptide has at least 6transmembrane domains (e.g., 6, 8, or 10 TM domains), and morepreferably, has at least 12 TM domains. The OCT-3 polypeptide can be amammalian polypeptide, i.e., a polypeptide normally expressed by thecells of a mammal, such as a human. In the event the OCT-3 polypeptideis human, it can have the sequence shown in SEQ ID NO:2 or SEQ ID NO:4,or it can be encoded by nucleic acid molecules having the sequence shownin SEQ ID NO:1 or SEQ ID NO:3. However, the invention is not limited tonucleic acid molecules and polypeptides that are identical to those SEQID Nos. For example, the invention includes nucleic acid molecules whichencode splice variants, allelic variants or mutant forms of OCT-3 aswell as the proteins encoded by such nucleic acid molecules. Also withinthe invention are nucleic acid molecules that hybridize under stringentconditions to a nucleic acid molecule having the sequence of SEQ ID NO:1or SEQ ID NO:3. As described further below, molecules that aresubstantially identical to SEQ ID Nos. 1-4 are also encompassed.

The term “substantially pure” as used herein in reference to a givencompound (e.g., an OCT-3 polypeptide) means that the compound issubstantially free from other compounds, such as those in cellularmaterial, viral material, or culture medium, with which the compound mayhave been associated (e.g., in the course of production by recombinantDNA techniques or before purification from a natural biological source).When chemically synthesized, a compound of the invention issubstantially pure when it is substantially free from the chemicalcompounds used in the process of its synthesis. Polypeptides or othercompounds of interest are substantially free from other compounds whenthey are within preparations that are at least 60% by weight (dryweight) the compound of interest. Preferably, the preparation is atleast 75%, more preferably at least 90%, and most preferably at least99%, by weight the compound of interest. Purity can be measured by anyappropriate standard method, for example, by column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

Where a particular polypeptide or nucleic acid molecule is said to havea specific percent identity to a reference polypeptide or nucleic acidmolecule of a defined length, the percent identity is relative to thereference polypeptide or nucleic acid molecule. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide which is 50% identical to the referencepolypeptide over its entire length. Of course, many other polypeptideswill meet the same criteria. The same rule applies for nucleic acidmolecules.

For polypeptides, the length of the reference polypeptide sequence willgenerally be at least 16 amino acids, preferably at least 20 aminoacids, more preferably at least 25 amino acids, and most preferably 35amino acids, 50 amino acids, or 100 amino acids. For nucleic acids, thelength of the reference nucleic acid sequence will generally be at least50 nucleotides, preferably at least 50 nucleotides, more preferably atleast 75 nucleotides, and most preferably at least 100 nucleotides(e.g., 150, 200, 250, or 300 nucleotides).

In the case of polypeptide sequences that are less than 100% identicalto a reference sequence, the non-identical positions are preferably, butnot necessarily, conservative substitutions for the reference sequence.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine.

Sequence identity can be measured using sequence analysis software(e.g., the Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705), with the default parameters as specifiedtherein.

The invention also features a host cell that includes an isolatednucleic acid molecule encoding OCT-3 (either alone or in conjunctionwith a heterologous polypeptide, such as a detectable marker), or anucleic acid vector that contains a sequence encoding OCT-3 (again, withor without a heterologous polypeptide). The vector can be an expressionvector, and can include a regulatory element.

An antibody that specifically binds an OCT-3 polypeptide is also withinthe scope of the present invention and is useful, for example, to detectOCT-3 in a biological sample, or to alter the activity of OCT-3. Forexample, OCT-3 can be detected in a biological sample by contacting thesample with an antibody that specifically binds OCT-3 under conditionsthat allow the formation of an OCT-3-antibody complex and detecting thecomplex, if present, as an indication of the presence of OCT-3 in thesample. The use of an antibody in a treatment regime, where it can alterthe activity of OCT-3, is discussed further below.

An antibody of the invention can be a monoclonal, polyclonal, orengineered antibody that specifically binds OCT-3 (as described morefully below). An antibody that “specifically binds” to a particularantigen, for example, an OCT-3 polypeptide of the invention, will notsubstantially recognize or bind to other molecules in a sample, such asa biological sample, that includes OCT-3.

Given that an object of the present invention is to alter the expressionor activity of OCT-3 in vivo, a pharmaceutical composition containing,for example, an isolated nucleic acid molecule encoding OCT-3 (or afragment thereof), a nucleic acid molecule that is antisense to OCT-3(i.e., that has a sequence that is the reverse and complement of aportion of the coding strand of an OCT-3 gene), an OCT-3 polypeptide, oran antibody, small molecule, or other compound that specifically bindsan OCT-3 polypeptide is also a feature of the invention.

The discovery and characterization of oct-3 and the polypeptide itencodes makes it possible to determine whether a given disorder isassociated with aberrent expression of oct-3 (meaning expression at thelevel of gene transcription or mRNA translation) or activity of OCT-3.For example, one can diagnose a patient as having a disorder associatedwith aberrant expression of oct-3 by measuring oct-3 expression in abiological sample obtained from the patient. An increase or decrease inoct-3 expression in the biological sample, compared with oct-3expression in a control sample (e.g., a sample of the same tissuecollected from one or more healthy individuals) indicates that thepatient has a disorder associated with aberrant expression of oct-3.Similarly, one can diagnose a patient as having a disorder associatedwith aberrant activity of OCT-3 by measuring OCT-3 activity in abiological sample obtained from the patient. An increase or decrease inOCT-3 activity in the biological sample, compared with OCT-3 activity ina control sample, indicates that the patient has a disorder associatedwith aberrant activity of OCT-3. The techniques required to measure geneexpression or polypeptide activity are well known to those of ordinaryskill in the art.

In addition to diagnostic methods, such as those described above, thepresent invention encompasses methods and compositions for typing andevaluating the prognosis, appropriate treatment, and treatmenteffectiveness of disorders associated with inappropriate expression ofoct-3 or inappropriate activity of OCT-3. For example, the nucleic acidmolecules of the invention can be used as probes to classify cells interms of their level of oct-3 expression, or as primers for diagnosticPCR analysis in which mutations, allelic variations, and regulatorydefects in the oct-3 gene can be detected. Similarly, those of ordinaryskill in the art can use routine techniques to identify inappropriateactivity of OCT-3, which can be observed in a variety of forms. Forexample, inappropriate activity can take the form of an alteration inthe rate with which an OCT-3 transporter moves molecules across theplasma membrane, or a difference in the type of molecule that istransported. Diagnostic kits for the practice of such methods are alsoprovided.

The invention further encompasses transgenic animals that express OCT-3and recombinant “knock-out” animals that fail to express OCT-3. Theseanimals can serve as new and useful models of disorders in which oct-3is misexpressed.

The invention also features antagonists and agonists of OCT-3 that caninhibit or enhance, respectively, one or more of the biologicalactivities of OCT-3. Suitable antagonists can include small molecules(i.e., molecules with a molecular weight below about 500), largemolecules (i.e., molecules with a molecular weight above about 500),antibodies that specifically bind and “neutralize” OCT-3 (as describedbelow), and nucleic acid molecules that interfere with transcription ortranslation of OCT-3 (e.g., antisense nucleic acid molecules andribozymes). Agonists of OCT-3 also include small and large molecules,and antibodies other than neutralizing antibodies.

The invention also features molecules that can increase or decrease theexpression of oct-3 (e.g., by altering transcription or translation).Small molecules (as defined above), large molecules (as defined above),and nucleic acid molecules (e.g., antisense and ribozyme molecules) canbe used to inhibit the expression of oct-3. Other types of nucleic acidmolecules (e.g., molecules that bind to oct-3 transcriptional regulatorysequences) can be used to increase the expression of oct-3.

Compounds that modulate the expression of oct-3 in a cell can beidentified by comparing the level of expression of oct-3 in the presenceof a selected compound with the level of expression of oct-3 in theabsence of that compound. A difference in the level of oct-3 expressionindicating that the selected compound modulates the expression of oct-3in the cell. A comparable test for compounds that modulate the activityof OCT-3 can be carried out by comparing the level of OCT-3 activity inthe presence and absence of the compound.

Patients who have a neurological disorder mediated by abnormal OCT-3activity can be treated by administration of a compound that alters theexpression of OCT-3 or the activity of OCT-3. When the objective is todecrease expression or activity, the compound administered can be anoct-3 antisense oligonucleotide or an antibody, such as a neutralizingantibody, that specifically binds OCT-3, respectively.

A wide variety of OCT-3 mediated neurological disorders are amenable totreatment according to the methods set forth herein. For example, theneurological disorder can be a neurodegenerative disease, such asParkinson's Disease, Huntington's Disease, or Alzheimer's Disease.Alternatively, the neurological disorder can be characterized byabnormal behavior or mood disorders. For example, the patient may sufferfrom depression, anxiety, or schizophrenia. A patient who has a sleepdisorder or a weight disorder, such as a patient who is obese or who issuffering from a wasting disorder (as often occurs in the event of AIDS)can also be treated by administration of a compound that alters theexpression of oct-3 or the activity of OCT-3. A detailed description ofthese methods of treatment is set forth below.

The preferred methods and materials are described below in exampleswhich are meant to illustrate, not limit, the invention. Skilledartisans will recognize methods and materials that are similar orequivalent to those described herein, and that can be used in thepractice or testing of the present invention.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the preferred methods andmaterials are described herein. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In addition, the materials, methods, andexamples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thedetailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C is a depiction of a human oct-3 nucleotide sequence(including 3′ and 5′ non-translated sequence; SEQ ID NO:1) that encodesan OCT-3 polypeptide with 12 transmembrane domains (SEQ ID NO:2).

FIGS. 2A-2B is a depiction of a human oct-3 nucleotide sequence(including 3′ and 5′ non-translated sequence; SEQ ID NO:3) that encodesan OCT-3 polypeptide with 6 TM domains (SEQ ID NO:4).

FIGS. 3A-3B is a depiction of a rat oct-3 nucleotide sequence (SEQ IDNO:5) encoding a rat OCT-3 polypeptide (SEQ ID NO:6). This polypeptidedoes not appear to be full-length.

FIGS. 4A-4E are representations of regions of amino acids in the OCT-3polypeptide (SEQ ID NO:2) that conform to consensus sequences derivedfrom polypeptides in the OCT family. Alternative residues for eachconsensus sequence are shown on the uppermost line. The sequences shownin FIG. 4A are found in the second transmembrane domain and continuebetween the second and third TM domains; the sequences shown in FIG. 4Bare found in the in the fourth transmembrane domain and continue betweenthe fourth and fifth TM domains; the sequences shown in FIG. 4C arefound between the sixth and seventh TM domains; the sequences shown inFIG. 4D are found C-terminal to the twelfth TM domain; and the sequencesshown in FIG. 4E are found in the tenth transmembrane domain andcontinue between the tenth and eleventh TM domains.

DETAILED DESCRIPTION

As described above, the nucleic acid molecules of the invention and thepolypeptides they encode (e.g., OCT-3 polypeptides or fragments thereof)can be used directly as diagnostic and therapeutic agents, or they canbe used to generate antibodies or identify small molecules that, inturn, are clinically useful. In addition, oct-3 nucleic acid moleculesare useful in genetic mapping, to identify the chromosomal location ofoct-3, and as tissue-specific (e.g., neuronal-specific) markers.Accordingly, expression vectors containing the nucleic acid molecules ofthe invention, cells transfected with these vectors, the polypeptidesexpressed, and antibodies generated, against either the entirepolypeptide or an antigenic fragment thereof, are among the preferredembodiments. These embodiments and their clinical application aredescribed further below.

I. Nucleic Acid Molecules Encoding OCT-3

The oct-3 nucleic acid molecules of the invention can be cDNA, genomicDNA, synthetic DNA, or RNA, and can be double-stranded orsingle-stranded. In the event the nucleic acid molecule issingle-stranded, it can be either a sense or an antisense strand.Fragments of these molecules are also considered within the scope of theinvention, and can be produced, for example, by the polymerase chainreaction (PCR), or generated by treating a longer fragment (e.g., afull-length oct-3 gene sequence) with one or more restrictionendonucleases. Similarly, a full-length oct-3 RNA molecule, or afragment thereof, can be produced by in vitro transcription. Theisolated nucleic acid molecule of the invention can encode a fragment ofOCT-3 that is not found as such in the natural state. Although nucleicacid molecules encoding any given fragment of OCT-3 are within the scopeof the invention, fragments that retain the biological activity of OCT-3(as assessed below) are preferred.

The nucleic acid molecules of the invention encompass recombinantmolecules, such as those in which a nucleic acid molecule (e.g., anisolated nucleic acid molecule encoding OCT-3, or a fragment thereof) isincorporated: (1) into a vector (e.g., a plasmid or viral vector), (2)into the genome of a heterologous cell, or (3) into the genome of ahomologous cell, at a position other than the natural chromosomallocation. Recombinant nucleic acid molecules, transgenic animals, anduses therefor are discussed further below.

The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide (e.g., the polypeptides of SEQ ID NO:2 or SEQ ID NO:4).In addition, the nucleic acid molecules of the invention are not limitedto those that encode the amino acid residues of the OCT-3 polypeptide asshown is SEQ ID NOs:1 and 2; they can also include some or all of thenon-coding sequences that lie upstream or downstream from an oct-3coding sequence, a heterologous regulatory element, or a sequenceencoding a heterologous polypeptide (e.g., a reporter gene). Regulatoryelements and reporter genes are discussed further below.

The nucleic acid molecules of the invention can be synthesized (forexample, by phosphoramidite-based synthesis) or obtained from abiological cell, such as the cell of a mammal. Thus, the nucleic acidscan be those of a human, mouse, rat, guinea pig, cow, sheep, goat,horse, pig, rabbit, monkey, dog, or cat. Combinations or modificationsof the nucleotides within these types of nucleic acid molecules are alsoencompassed.

In the event the nucleic acid molecules of the invention encode or actas antisense molecules, they can be used, for example, to regulatetranslation of oct-3 mRNA. Techniques associated with detection ofnucleic acid sequences or regulation of their expression are well knownto persons of ordinary skill in the art, and can be used in the contextof the present invention to diagnose or treat disorders associated withaberrant oct-3 expression. However, aberrent expression of oct-3 (oraberrent activity of OCT-3) is not a prerequisite for treatmentaccording to the methods of the invention; the molecules of theinvention (including the nucleic acid molecules described here) areexpected to be useful in improving the symptoms associated with avariety of medical conditions (particularly neurological disorders)regardless of whether or not the expression of oct-3 (or the activity ofOCT-3) is detectably aberrent. Nucleic acid molecules are discussedfurther below in the context of their clinical utility.

The invention also encompasses nucleic acid molecules that encode othermembers of the OCT-3 family. Such nucleic acid molecules will be readilyidentified by the ability to hybridize under stringent conditions to anucleic acid molecule encoding an OCT-3 polypeptide (SEQ ID NOs:1 and3). The cDNA sequences described herein (SEQ ID NOs:1, 3, and 5) can beused to identify these nucleic acids, which include, for example,nucleic acids that encode homologous polypeptides in other species,splice variants of the oct-3 gene in humans or other mammals, allelicvariants of the oct-3 gene in humans or other mammals, and mutant formsof the oct-3 gene in humans or other mammals.

The preferred class of nucleic acid molecules that hybridize to SEQ IDNOs:1 and 3 are nucleic acid molecules that encode human allelicvariants of OCT-3. There are two major classes of such variants: activeallelic variants, naturally occurring variants of SEQ ID NOs:2 and 4that have the ability to act as a transporter and non-active allelicvariants, naturally occurring allelic variants of SEQ ID NOs:2 and 4that are unable to function as a transporter. Active allelic variantscan be used as an equivalent for an OCT-3 protein having the amino acidsequence of SEQ ID NOs:2 or 4 as described herein whereas nonactiveallelic variants can be used in methods of disease diagnosis and as atherapeutic target.

The invention features methods of detecting and isolating such nucleicacid molecules. Using these methods, a sample (e.g., a nucleic acidlibrary, such as a cDNA or genomic library) is contacted (or “screened”)with an oct-3-specific probe (e.g., a fragment of SEQ ID NO:1 that is atleast 17 nucleotides long). The probe will selectively hybridize tonucleic acids encoding related polypeptides (or to complementarysequences thereof). The term “selectively hybridize” is used to refer toan event in which a probe binds to nucleic acid molecules encoding OCT-3(or to complementary sequences thereof) to a detectably greater extentthan to nucleic acids encoding other polypeptides, particularly othertypes of transporter molecules (or to complementary sequences thereof).The probe, which can contain at least 17 nucleotides (e.g., 18, 20, 25,50, 100, 150, or 200 nucleotides) can be produced using any of severalstandard methods (see, e.g., Ausubel et al., “Current Protocols inMolecular Biology, Vol. I,” Green Publishing Associates, Inc., and JohnWiley & Sons, Inc., N.Y., 1989). For example, the probe can be generatedusing PCR amplification methods in which oligonucleotide primers areused to amplify an oct-3-specific nucleic acid sequence (for example, anucleic acid encoding one of the transmembrane domains) that can be usedas a probe to screen a nucleic acid library and thereby detect nucleicacid molecules (within the library) that hybridize to the probe.

One single-stranded nucleic acid is said to hybridize to another if aduplex forms between them. This occurs when one nucleic acid contains asequence that is the reverse and complement of the other (this samearrangement gives rise to the natural interaction between the sense andantisense strands of DNA in the genome and underlies the configurationof the double helix). Complete complementarity between the hybridizingregions is not required in order for a duplex to form; it is onlynecessary that the number of paired bases is sufficient to maintain theduplex under the hybridization conditions used.

Typically, hybridization conditions initially used to identify relatedgenes are of low to moderate stringency. These conditions favor specificinteractions between completely complementary sequences, but allow somenon-specific interaction between less than perfectly matched sequencesto occur as well. After hybridization, the nucleic acids can be “washed”under moderate or high conditions of stringency to dissociate duplexesthat are bound together by some non-specific interaction (the nucleicacids that form these duplexes are thus not completely complementary).

As is known in the art, the optimal conditions for washing aredetermined empirically, often by gradually increasing the stringency.The parameters that can be changed to affect stringency include,primarily, temperature and salt concentration. In general, the lower thesalt concentration and the higher the temperature, the higher thestringency. Washing can be initiated at a low temperature (e.g., roomtemperature) using a solution containing a salt concentration Chat isequivalent to or lower than that of the hybridization solution.Subsequent washing can be carried out using progressively warmersolutions having the same salt concentration. As alternatives, the saltconcentration can be lowered and the temperature maintained in thewashing step, or the salt concentration can be lowered and thetemperature increased. Additional parameters can also be altered. Forexample, use of a destabilizing agent, such as formamide, alters thestringency conditions.

In reactions where nucleic acids are hybridized, the conditions used toachieve a given level of stringency will vary. There is not one set ofconditions, for example, that will allow duplexes to form between allnucleic acids that are 85% identical to one another; hybridization alsodepends on unique features of each nucleic acid. The length of thesequence, the composition of the sequence (e.g., the content ofpurine-like nucleotides versus the content of pyrimidine-likenucleotides) and the type of nucleic acid (e.g., DNA or RNA) affecthybridization. An additional consideration is whether one of the nucleicacids is immobilized (e.g., on a filter).

An example of a progression from lower to higher stringency conditionsUs the following, where the salt content is given as the relativeabundance of SSC (a salt solution containing sodium chloride and sodiumcitrate; 2×SSC is 10-fold more concentrated than 0.2×SSC). Nucleic acidmolecules are hybridized at 42° C. in 2×SSC/0.1% SDS (sodiumdodecylsulfate; a detergent) and then washed in 0.2×SSC/0.1% SDS at roomtemperature (for conditions of low stringency); 0.2×SSC/0.1% SDS at 42°C. (for conditions of moderate stringency); and 0.1×SSC at 68° C. (forconditions of high stringency). Washing can be carried out using onlyone of the conditions given, or each of the conditions can be used (forexample, washing for 10-15 minutes each in the order listed above). Anyor all of the washes can be repeated. As mentioned above, optimalconditions will vary and can be determined empirically.

A second set of conditions that are considered “stringent conditions”are those in which hybridization is carried out at 50° C. in Churchbuffer (7% SDS, 0.5% NaHPO₄, 1 M EDTA, 1% BSA) and washing is carriedout at 50° C. in 2×SSC.

Preferably, nucleic acid molecules of the invention that are defined bytheir ability to hybridize with SEQ ID Nos. 1, 3, or 5 under stringentconditions will have additional features in common with oct-3. Forexample, the nucleic acid molecules identified by hybridization may havea similar, or identical, expression profile as the oct-3 moleculesdescribed herein, or may encode a polypeptide having one or more of thebiological activities possessed by OCT-3.

Once detected, the nucleic acid molecules can be isolated by any of anumber of standard techniques (see, e.g., Sambrook et al., “MolecularCloning, A Laboratory Manual,” 2nd Ed. Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989).

The invention also encompasses: (a) expression vectors that contain anyof the foregoing oct-3 or oct-3-related coding sequences and/or theircomplements (i.e., “antisense” sequence) and fragments thereof; (b)expression vectors that contain any of the foregoing oct-3-relatedsequences operatively associated with a regulatory element (examples ofwhich are given below) that directs the expression of the codingsequences; (c) expression vectors containing, in addition to sequencesencoding an OCT-3 polypeptide, nucleic acid sequences that are unrelatedto nucleic acid sequences encoding OCT-3, such as molecules encoding areporter or marker; and (d) genetically engineered host cells thatcontain any of the foregoing expression vectors, and thereby express thenucleic acid molecules of the invention in the host cell. The regulatoryelements referred to above include, but are not limited to, inducibleand non-inducible promoters, enhancers, operators and other elements,which are known to those skilled in the art, and which drive orotherwise regulate gene expression. Such regulatory elements include butare not limited to the cytomegalovirus hCMV immediate early gene, theearly or late promoters of SV40 adenovirus, the lac system, the trpsystem, the TAC system, the TRC system, the major operator and promoterregions of phage λ, the control regions of fd coat protein, the promoterfor 3-phosphoglycerate kinase, the promoters of acid phosphatase, andthe promoters of the yeast α-mating factors.

Additionally, the OCT-3 encoding nucleic acid molecules of the presentinvention can form part of a hybrid gene encoding additional polypeptidesequences, for example, sequences that function as a marker or reporter.Examples of marker or reporter genes include β-lactamase,chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA),aminoglycoside phosphotransferase (neo^(r), G418^(r)) dihydrofolatereductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidinekinase (TK), lacz (encoding β-galactosidase), and xanthine guaninephosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional useful reagents, for example, ofadditional sequences that can serve the function of a marker orreporter. Generally, a chimeric or hybrid polypeptide of the inventionwill include a first portion and a second portion; the first portionbeing an OCT-3 polypeptide or a fragment thereof (preferably abiologically active fragment) and the second portion being, for example,the reporter described above or an immunoglobulin constant region.

The expression systems that can be used for purposes of the inventioninclude, but are not limited to, microorganisms such as bacteria (e.g.,E. coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA, or cosmid DNA expression vectors containing the nucleicacid molecules of the invention; yeast (e.g., Saccharomyces and Pichia)transformed with recombinant yeast expression vectors containing thenucleic acid molecules of the invention (preferably containing a nucleicacid sequence encoding human OCT-3 (such as the sequence of SEQ ID NO:1or SEQ ID NO:3)); insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing a nucleic acidmolecule of the invention; plant cell systems infected with recombinantvirus expression vectors (e.g., cauliflower mosaic virus (CaMV) andtobacco mosaic virus (TMV)) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing oct-3 nucleotidesequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, VERO,HeLa, MDCK, WI38, and NIH 3T3 cells) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., the metallothionein promoter) or from mammalian viruses(e.g., the adenovirus late promoter and the vaccinia virus 7.5Kpromoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the geneproduct being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions containing OCT-3 polypeptides or for raising antibodies tothose polypeptides, vectors that are capable of directing the expressionof high levels of fusion protein products that are readily purified maybe desirable. Such vectors include, but are not limited to, the E. coliexpression vector pUR278 (Ruther et al., EMBO J. 2:1791, 1983), in whichthe coding sequence of the insert may be ligated individually into thevector in frame with the lacZ coding region so that a fusion protein isproduced; pIN vectors (Inouye and Inouye, Nucleic Acids Res.13:3101-3109, 1985; Van Heeke and Schuster, J. Biol. Chem.264:5503-5509, 1989); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption toglutachione-agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhidrosis virus(AcNPV) can be used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The coding sequence of the insertmay be cloned individually into non-essential regions (e.g., thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (e.g., the polyhedrin promoter). Successful Insertion of thecoding sequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed (e.g., see Smith et al., J. Virol. 46:584,1983; and Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the nucleic acid molecule of the invention can be ligated to anadenovirus transcription/translation control complex, for example, thelate promoter and tripartite leader sequence. This chimeric gene maythen be inserted in the adenovirus genome by in vitro or in vivorecombination. Insertion in a non-essential region of the viral genome(e.g., region E1 or E3) will result in a recombinant virus that isviable and capable of expressing an oct-3 gene product in infected hosts(e.g., see Logan and Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659,1984). Specific initiation signals may also be required for efficienttranslation of inserted nucleic acid molecules. These signals includethe ATG initiation codon and adjacent sequences. In cases where anentire gene or cDNA, including its own initiation codon and adjacentsequences, is inserted into the appropriate expression vector, noadditional translational control signals may be needed. However, incases where only a portion of the coding sequence is inserted (e.g., theportion encoding the mature form of an OCT-3 protein) endoexogenoustranslational control signals, including, perhaps, the ATG initiationcodon, must be provided. Furthermore, the initiation codon must be inphase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see Bittner et al., Methods in Enzymol.153:516-544, 1987).

In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. The mammalian celltypes listed above are among those that could serve as suitable hostcells.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe OCT-3 sequences described above can be engineered. Rather than usingexpression vectors that contain viral origins of replication, host cellscan be transformed with DNA controlled by appropriate expression controlelements (e.g., promoter sequences, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells can beallowed to grow for 1-2 days in an enriched media, and then switched toa selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection, and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form fociwhich, in turn, can be cloned and expanded into cell lines. This methodcan advantageously be used to engineer cell lines that express OCT-3.Such engineered cell lines may be particularly useful in screening andevaluating compounds that affect the endogenous activity of the geneproduct (i.e., OCT-3).

A number of selection systems can be used. For example, the herpessimplex virus thymidine kinase (Wigler, et al., Cell 11:223, 1977),hypoxanthine-guanine phosphoribosyltransferase (Szybalska and Szybalski,Proc. Natl. Acad. Sci. USA 48:2026, 1962), and adeninephosphoribosyltransferase (Lowy, et al., Cell 22:817, 1980) genes can beemployed in tk, hgprt or aprt cells, respectively. Also, anti-metaboliteresistance can be used as the basis of selection for the followinggenes: dhfr, which confers resistance to methotrexate (Wigler et al.,Proc. Natl. Acad. Sci. USA 77:3567, 1980; O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527, 1981); gpt, which confers resistance tomycophenolic acid (Mulligan and Berg, Proc. Natl. Acad. Sci. USA78:2072, 1981); neo, which confers resistance to the aminoglycosideG-418 (Colberre-Garapin et al., J. Mol. Biol. 150:1, 1981); and hygro,which confers resistance to hygromycin (Santerre et al., Gene 30:147,1984).

Alternatively, any OCT-3-containing fusion protein can be readilypurified by utilizing an antibody specific or the fusion protein beingexpressed. For example, a system described by Janknecht et al. allowsfor the ready purification of non-denatured fusion proteins expressed inhuman cell lines (Proc. Natl. Acad. Sci. USA 88:8972-8976, 1991). Inthis system, the gene of interest is subcloned into a vacciniarecombination plasmid such that the gene's open reading frame istranslationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺ nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

As implied by the descriptions above, a host cell is any cell into which(or into an ancestor of which) a nucleic acid encoding a polypeptide ofthe invention (e.g., an OCT-3 polypeptide) has been introduced by meansof recombinant DNA techniques.

II. OCT-3 Polypeptides

The OCT-3 polypettides described herein are those encoded by any of thenucleic acid molecules described above, and include fragments of OCT-3,mutant forms of OCT-3, active and non-active allelic variants of OCT-3,splice variants of OCT-3, truncated forms of OCT-3, and fusion proteinscontaining all or a portion of OCT-3. These polypeptides can be preparedfor a variety of uses including, but not limited to, the generation ofantibodies, as reagents in diagnostic assays, for the identification ofother cellular gene products or exogenous compounds that can modulatethe activity or expression of OCT-3, and as pharmaceutical reagentsuseful for the treatment of any disorder in which the associatedsymptoms are improved by altering the activity of OCT-3.

The terms “protein” and “polypeptide” are used herein to describe anychain of amino acid residues, regardless of length or post-translationalmodification (e.g., modification by glycosylation or phosphorylation).Thus, the term “OCT-3 polypeptide” includes full-length, naturallyoccurring OCT-3 polypeptides (that can be purified from tissues in whichthey are naturally expressed, according to standard biochemical methodsof purification), as well as recombinantly or synthetically producedpolypeptides that correspond either to a full-length,naturally-occurring OCT-3 polypeptide or to particular domains orportions of such a polypeptide. The term also encompasses mature OCT-3having an added amino-terminal methionine (useful for expression inprokaryotic cells).

Preferred polypeptides are substantially pure OCT-3 polypeptides thatare at least 50% (e.g., 55%, 60%, or 65%), more preferably at least 70%(e.g., 72%, 75%, or 78%), even more preferably at least 80% (e.g., 80%,85% or 90%), and most preferably at least 95% (e.g., 97% or even 99%)identical to SEQ ID NO:2 or SEQ ID NO:4. Those of ordinary skill in theart are well able to determine the percent identity between two aminoacid sequences. Further guidance on this point is provided above. Inaddition, in FIGS. 4A-4E, regions of amino acid sequence that, alongwith the TM domains, are conserved between OCT-3 and other members ofthe OCT family of proteins (OCT-1 and OCT-2) are shown. Thus, if apolypeptide is encoded by a nucleic acid that hybridizes under stringentconditions with the oct-3 sequence disclosed herein and also encodes oneor more of the conserved regions present in OCT-3, it will be recognizedas an OCT-3 polypeptide and thereby considered within the scope of thepresent invention.

The invention also encompasses polypeptides that are functionallyequivalent to OCT-3. These polypeptides are equivalent to OCT-3 in thatthey are capable of carrying out one or more of the functions of OCT-3in a biological system. Polypeptides that are functionally equivalent toOCT-3 can have 20%, 40%, 50%, 75%, 80%, or even 90% of one or more ofthe biological activities of the full-length, mature human form ofOCT-3. Such comparisons are generally based on an assay of biologicalactivity in which equal concentrations of the polypeptides (i.e., anOCT-3 polypeptide disclosed herein and a candidate OCT-3 polypeptide)are used and compared. The comparison can also be based on the amount ofthe polypeptide required to reach 50% of the maximal biological activityobtainable.

Functionally equivalent proteins can be those, for example, that containadditional or substituted amino acid residues. Substitutions may be madeon the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. Amino acids that are typically considered to providea conservative substitution for one another are specified in the Summaryof the Invention.

Polypeptides that are functionally equivalent to OCT-3 (SEQ ID NO:2) canbe made using random mutagenesis techniques well known to those ofordinary skill in the art (and the resulting mutant OCT-3 polypeptidescan be tested or activity). It is more likely, however, that suchpolypeptides will be generated by site-directed mutagenesis (again usingtechniques well known to persons of ordinary skill in the art). Thesepolypeptides may have increased functionality or decreasedfunctionality.

To design functionally equivalent polypeptides, it is useful todistinguish between conserved positions and variable positions. This canbe done by aligning the sequence of oct-3 cDNAs that were obtained fromvarious organisms. Skilled artisans will recognize that conserved aminoacid residues are more likely to be necessary for preservation offunction. Thus, it is preferable that conserved residues are notaltered.

Mutations within the oct-3 coding sequence can be made to generatevariant oct-3 genes that are better suited for expression in a selectedhost cell. For example, N-linked glycosylation sites can be altered oreliminated to achieve, for example, expression of a homogeneous productthat is more easily recovered and purified from yeast hosts which areknown to hyperglycosylate N-linked sites. To this end, a variety ofamino acid substitutions at one or both of the first or third amino acidpositions of any one or more of the glycosylation recognition sequenceswhich occur (in N-X-S or N-X-), and/or an amino acid deletion at thesecond position of any one or more of such recognition sequences, willprevent glycosylation at the modified tripeptide sequence (see, e.g.,Miyajima et al., EMBO J. 5:1193, 1986).

The polypeptides of the invention can be expressed fused to anotherpolypeptide, for example, a marker polypeptide or fusion partner. Forexample, the polypeptide can be fused to a hexa-histidine tag tofacilitate purification of bacterially expressed protein or ahemagglutinin tag to facilitate purification of protein expressed ineukaryotic cells. In addition, an OCT-3 polypeptide can be fused to GST.

The polypeptides of the invention can be chemically synthesized (e.g.,see Creighton, “Proteins: Structures and Molecular Principles,” W. H.Freeman & Co., N.Y., 1983), or, perhaps more advantageously, produced byrecombinant DNA technology as described herein. For additional guidance,persons of ordinary skill in the art may consult Ausubel et al. (supra),Sambrook et al. (“Molecular Cloning, A Laboratory Manual,” Cold SpringHarbor Press, Cold Spring Harbor, N.Y., 1989), and, particularly forexamples of chemical synthesis, Gait, M. J. (“Oligonucletide Synthesis,”IRL Press, Oxford, 1984).

The invention also features polypeptides that interact with OCT-3 (andthe genes that encode them) and thereby alter the function or activityof OCT-3. Interacting polypeptides can be identified using methods knownto those of ordinary skill in the art. One suitable method is the“two-hybrid system,” which detects protein interactions in vivo (Chienet al., Proc. Natl. Acad. Sci. USA, 88:9578, 1991). A kit for practicingthis method is available from Clontech (Palo Alto, Calif.).

III. Transgenic Animals

OCT-3 polypeptides can also be expressed in transgenic animals. Suchtransgenic animals represent model systems for the study of disordersthat are either caused by or exacerbated by misexpression of oct-3, ordisorders that can be treated by altering the expression of oct-3 or theactivity of OCT-3 (even though the expression or activity is notdetectably abnormal). Transgenic animals can also be used for thedevelopment of therapeutic agents that modulate the expression of oct-3or the activity of OCT-3.

Transgenic animals can be farm animals (e.g., pigs, goats, sheep, cows,horses, rabbits, and the like) rodents (such as rats, guinea pigs, andmice), non-human primates (e.g., baboons, monkeys, and chimpanzees), anddomestic animals (e.g., dogs and cats). Transgenic mice are especiallypreferred.

Any technique known in the art can be used to introduce an oct-3transgene into animals to produce founder lines of transgenic animals.Such techniques include, but are not limited to, pronuclearmicroinjection (U.S. Pat. No. 4,873,191); retrovirus mediated genetransfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci.,USA 82:6148, 1985); gene targeting into embryonic stem cells (Thompsonet al., Cell 56:313, 1989); and electroporation of embryos (Lo, Mol.Cell. Biol. 3:1803, 1983).

The present invention provides for transgenic animals that carry anoct-3 transgene in all of their cells, as well as animals that carry atransgene in some, but not all of their cells. For example, theinvention provides for mosaic animals. The oct-3 transgene can beintegrated as a single transgene or in concatamers, for example,head-to-head tandems or head-to-tail tandems. The transgene can also beselectively introduced into, and activated in, a particular cell type(Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232, 1992). The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art.

When it is desired that an oct-3 transgene be integrated into thechromosomal site of an endogenous oct-3 gene, gene targeting ispreferred. Briefly, when such a technique is to be used, vectorscontaining some nucleotide sequences homologous to an endogenous oct-3gene are designed for the purpose of integrating, via homologousrecombination with chromosomal sequences, into and disrupting thefunction of the nucleotide sequence of the endogenous gene. Thetransgene also can be selectively introduced into a particular celltype, thus inactivating the endogenous oct-3 gene in only that cell type(Gu et al., Science 265:103, 1984). The regulatory sequences requiredfor such a cell-type specific inactivation will depend upon theparticular cell type of interest, and will be apparent to those of skillin the art. These techniques are useful for preparing “knock outs”having no functional oct-3 gene.

Once transgenic animals have been generated, the expression of therecombinant oct-3 gene can be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to determine whether integration of the transgene has takenplace. The level of mRNA expression of the transgene in the tissues ofthe transgenic animals may also be assessed using techniques whichinclude, but are not limited to, Northern blot analysis of tissuesamples obtained from the animal, in situ hybridization analysis, andRT-PCR. Samples of oct-3 gene-expressing tissue can also be evaluatedimmunocytochemically using antibodies specific for the OCT-3 transgeneproduct.

For a review of techniques that can be used to generate and assesstransgenic animals, those of ordinary skill in the art can consultGordon (Intl. Rev. Cytol. 115:171-229, 1989), and may obtain additionalguidance from, for example: Hogan et al. “Manipulating the Mouse Embryo”(Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1986; Krimpenfortet al., Bio/Technology 9:86, 1991; Palmiter et al., Cell 41:343, 1985;Kraemer et al., “Genetic Manipulation of the Early Mammalian Embryo,”Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1985; Hammer et al.,Nature 315:680, 1985; Purcel et al., Science 244:1281, 1986; Wagner etal., U.S. Pat. No. 5,175,385; and Krimpenfort et al., U.S. Pat. No.5,175,384.

The transgenic animals of the invention can be used to determine theconsequence of altering the expression of oct-3 in the context ofvarious disease states. For example, oct-3 knock out mice can begenerated using an established line of mice that serve as a model for aneurodegenerative disease. For example, oct-3 can be knocked out in micebearing a mutation in the weaver gene, which allows them to serve as amodel for Parkinson's Disease. If the symptoms (e.g., altered balance orgait) normally apparent in weaver mice are lessened when an oct-3 geneis no longer expressed, patients suffering from Parkinson's Disease arelikely to benefit from treatment with any compound that decreases theexpression of oct-3 or the activity of OCT-3.

IV. Anti-OCT-3 Antibodies

OCT-3 polypeptides (or immunogenic fragments or analogs thereof) can beused to raise antibodies useful in the invention; such polypeptides canbe produced by recombinant techniques or synthesized (see, for example,“Solid Phase Peptide Synthesis,” supra; Ausubel et al., supra). Ingeneral, OCT-3 polypeptides can be coupled to a carrier protein, such asKLH, as described in Ausubel et al., supra, mixed with an adjuvant, andinjected into a host mammal. Antibodies produced in that animal can thenbe purified by pepotide antigen affinity chromatography.

In particular, various host animals can be immunized by injection with aOCT-3 polypeptide or an antigenic fragment thereof. Commonly employedhost animals include rabbits, mice, guinea pigs, and rats. Variousadjuvants that can be used to increase the immunological response dependon the host species and include Freund's adjuvant (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, and dinitrophenol. Potentiallyuseful human adjuvants include BCG (bacille Calmette-Guerin) andCorynebacterium parvum. Polyclonal antibodies are heterogeneouspopulations of antibody molecules that are contained in the sera of theimmunized animals.

Antibodies within the invention therefore include polyclonal antibodiesand, in addition, monoclonal antibodies, humanized or chimericantibodies, single chain antibodies, Fab fragments, F(ab′)₂ fragments,and molecules produced using a Fab expression library.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, can be prepared using the OCT-3 polypeptidesdescribed above and standard hybridoma technology (see, for example,Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol.6:511, 1976; Kohler et al., Eur. J. Immunol. 6:292, 1976; Hammerling etal., “Monoclonal Antibodies and T Cell Hybridomas,” Elsevier, N.Y.,1981; Ausubel et al., supra).

In particular, monoclonal antibodies can be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture such as described in Kohler et al., Nature256:495, 1975, and U.S. Pat. No. 4,376,110; the human B-cell hybridomatechnique (Kosbor et al., Immunology Today 4:72, 1983; Cole et al.,Proc. Natl. Acad. Sci. USA 80:2026, 1983), and the EBV-hybridomatechnique (Cole et al., “Monoclonal Antibodies and Cancer Therapy,” AlanR. Liss, Inc., pp. 77-96, 1983). Such antibodies can be of anyimmunoglobulin class including pG, IgM, IgE, IgA, IgD and any subclassthereof. The hybridoma producing the mAb of this invention may becultivated in vitro or in vivo. The ability to produce high titers ofmAbs in vivo makes this a particularly useful method of production.

Once produced, polyclonal or monoclonal antibodies are tested forspecific OCT-3 recognition by Western blot or immunoprecipitationanalysis by standard methods, for example, as described in Ausubel etal., supra. Antibodies that specifically recognize and bind to OCT-3 areuseful in the invention. For example, such antibodies can be used in animmunoassay to monitor the level of OCT-3 produced by a mammal (e.g., todetermine the amount or subcellular location of OCT-3).

There are two major classes of antibodies which are within the scope ofthe present invention. The first class are antibodies that selectivelybind to OCT-3 polypeptide, not bind to other members of the OCT familyof proteins. The second class are antibodies that bind to more than onemember of the OCT family of proteins.

Preferably, OCT-3 selective antibodies of the invention are producedusing fragments of the OCT-3 polypeptide that lie outside highlyconserved regions (such as the TM domains and the regions shown in FIGS.4A-4E) and appear likely to be antigenic, by criteria such as highfrequency of charged residues. Cross-reactive anti-OCT-3 antibodies areproduced using a fragment of OCT-3 that is conserved amongst members ofthis family of proteins. in one specific example, such fragments aregenerated by standard techniques of PCR, and are then cloned into thepGEX expression vector (Ausubel et al., supra). Fusion proteins areexpressed in E. coli and purified using a glutathione agarose affinitymatrix as described in Ausubel, et al., supra.

In some cases it may be desirable to minimize the potential problems oflow affinity or specificity of antisera. In such circumstances, two orthree fusions can be generated for each protein, and each fusion can beinjected into at least two rabbits. Antisera can be raised by injectionsin a series, preferably including at least three booster injections.

Antiserum is also checked for its ability to immunoprecipitaterecombinant OCT-3 polypeptides or control proteins, such asglucocorticoid receptor, CAT, or luciferase.

The antibodies can be used, for example, in the detection of OCT-3 in abiological sample as part of a diagnostic assay or to reduce OCT-3activity as part of a theraputic regime (e.g., to reduce an undesirablyhigh level of OCT-3 activity). Antibodies also can be used in ascreening assay to measure the effect of a candidate compound onexpression or localization of OCT-3. Additionally, such antibodies canbe used in conjunction with the gene therapy techniques. For example,they may be used to evaluate the normal and/or engineeredOCT-3-expressing cells prior to their introduction into the patient.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1984;Neuberger et al., Nature 312:604, 1984; Takeda et al., Nature 314:452,1984) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine mAb and a human immunoglobulin constant region.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. Nos. 4,946,778, 4,946,778, and 4,704,692) can beadapted to produce single chain antibodies against an OCT-3 polypeptide,or a fragment thereof. Single chain antibodies are formed by linking theheavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain polypeptide.

Antibody fragments that recognize and bind to specific epitopes can begenerated by known techniques. For example, such fragments include butare not limited to F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed (Huse et al.,Science 246:1275, 1989) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Ant-bodies can be humanized by methods known in the art. For example,monoclonal antibodies with a desired binding specificity can becommercially humanized (Scotgene, Scotland; Oxford Molecular, Palo Alto,Calif.). Fully human antibodies, such as those expressed in transgenicanimals are also features of the invention (Green et al., NatureGenetics 7:13-21, 1994; see also U.S. Pat. Nos. 5,545,806 and5,569,825).

The methods described herein, in which anti-OCT-3 antibodies areemployed, can be performed, for example, by utilizing pre-packageddiagnostic kits comprising at least one specific oct-3 nucleotidesequence or antibody reagent described herein, which may be convenientlyused, for example, in clinical settings, to diagnose patients exhibitingsymptoms of the disorders described below.

V. Antisense Nucleic Acid Molecules

Treatment regimes based on an “antisense” approach involve the design ofoligonucleotides (either DNA or RNA) that are complementary to a portionof a selected mRNA. These oligonucleotides bind to complementary mRNAtranscripts and prevent their translation. Absolute complementarity,although preferred, is not required. A sequence “complementary” to aportion of an RNA molecule, as referred to herein, is a sequence havingsufficient complementarily to hybridize with the RNA, forming a stableduplex; in the case of double-stranded antisense nucleic acids, a singlestrand of the duplex DNA can be tested, or triplex formation can beassayed. The ability to hybridize will depend on both the degree ofcomplementarily and the length of the antisense nucleic acid. Generally,the longer the hybridizing nucleic acid, the more base mismatches withan RNA it may contain and still form a stable duplex (or triplex, as thecase may be). One of ordinary skill in the art can ascertain a tolerabledegree of mismatch by use of standard procedures to determine themelting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,for example, the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs recently have been shown to be effective atinhibiting translation of mRNAs as well (Wagner, Nature 372:333, 1984).Thus, oligonucleotides complementary to either the 5′ or 3′non-translated, non-coding regions of an oct-3 gene, for example, ahuman gene as shown in FIGS. 1A-1C or FIGS. 2A-2B, could be used in anantisense approach to inhibit translation of endogenous oct-3 mRNA.Oligonucleotides complementary to the 5′ untranslated region of the mRNAshould include the complement of the AUG start codon.

Antisense oligonucleotides complementary to mRNA coding regions are lessefficient inhibitors of translation but could be used in accordance withthe invention. Whether designed to hybridize to the 5′, 3′, or codingregion of oct-3 mRNA, antisense nucleic acids should be at least sixnucleotides in length, and are preferably oligonucleotides ranging from6 to about 50 nucleotides in length. In specific aspects, theoligonucleotide is at least 10 nucleotides, at least 17 nucleotides, atleast 25 nucleotides, or at least 50 nucleotides.

Regardless of the choice of target sequence, as with other therapeuticstrategies directed to OCT-3, it is preferred that in vitro studies arefirst performed to assess the ability of an antisense oligonucleotide toinhibit gene expression. If desired, the assessment can be quantitative.It is preferred that these studies utilize controls that distinguishbetween antisense gene inhibition and any nonspecific biological effectthat an oligonucleotide may incur. It is also preferred that thesestudies compare levels of the target RNA or protein with that of aninternal control RNA or protein. Additionally, it is envisioned thatresults obtained using an antisense oligonucleotide are compared withthose obtained using a control oligonucleotide. Preferably, the controloligonucleotide is of approximately the same length as the testoligonucleotide, and the nucleotide sequence of the controloligonucleotide differs from that of the test antisense sequence no morethan is necessary to prevent specific hybridization between the controloligonucleotide and the targeted RNA sequence.

The oligonucleotides can contain DNA or RNA, or they can containchimeric mixtures, derivatives, or modified versions thereof that areeither single-stranded or double-stranded. The oligonucleotide can bemodified at the base moiety, sugar moiety, or phosphate backbone, forexample, to improve stability of the molecule, hybridization, etc.Modified sugar moieties can be selected from the group including, butnot limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose. Amodified phosphate backbone can be selected from the group consisting ofa phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal, or an analog of any of thesebackbones.

The oligonucleotide can include other appended groups such as peptides(e.g., for disrupting the transport properties of the molecule in hostcells in vivo), or agents that facilitate transport across the cellmembrane (as described, for example, in Letsinger et al., Proc. Natl.Acad. Sci. USA 86:6553, 1989; Lemaitre et al., Proc. Natl. Acad. Sci.USA 84:648, 1987; PCT Publication No. WO 88/09810) or the blood-brainbarrier (see, for example, PCT Publication No. WO 89/10134), orhybridization-triggered cleavage agents (see, for example, Krol et al.,BioTechniques 6:958, 1988), or intercalating agents (see, for example,Zon, Pharm. Res. 5:539, 1988). To this end, the oligonucleotide can beconjugated to another molecule, for example, a peptide, a hybridizationtriggered cross-linking agent, a transport agent, or ahybridization-triggered cleavage agent.

An antisense oligonucleotide of the invention can comprise at least onemodified base moiety that is selected from the group including, but notlimited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethyl-aminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-theouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 2-(3-amino-3-N-2-carboxypropl) uracil, (acp3)w,and 2,6-diaminopurine.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oliaonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,Nucl. Acids. Res. 15:6625, 1987). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131,1987), or a chimeric RNA-DNA analog (Inoue et al., FEBS Lett. 215:327,1987).

Antisense oligonucleotides of the invention can be synthesized bystandard methods known in the art, for example, by use of an automatedDNA synthesizer (such as are commercially available from Biosearch,Applied Biosystems, etc.). As examples, phosphorothioateoligonucleotides can be synthesized by the method of Stein et al. (Nucl.Acids Res. 16:3209, 1988), and methylphosphonate oligonucleotides can beprepared by use of controlled pore glass polymer supports (Sarin et al.,i Proc. Natl. Acad. Sci. USA 85:7448, 1988).

While antisense oligonucleotides that are complementary to the codingregion of oct-3 could be used, those complementary to the transcribeduntranslated region are most preferred.

For therapeutic application, antisense molecules of the invention shouldbe delivered to cells that express oct-3 in vivo. A number of methodshave been developed for delivering antisense DNA or RNA to cells; forexample, antisense molecules can be injected directly into the tissuesite. Alternatively, modified antisense molecules, which are designed totarget cells that express oct-3 (e.g., antisense molecules linked topeptides or antibodies that specifically bind receptors or antigensexpressed on the target cell surface) can be administered systemically.

However, it is often difficult to achieve intracellular concentrationsof antisense molecules that are sufficient to suppress translation ofendogenous mRNAs. Therefore, a preferred approach uses a recombinant DNAconstruct in which the antisense oligonucleotide is placed under thecontrol of a strong pol III or pol II promoter. The use of such aconstruct to transfect target cells in the patient will result in thetranscription of sufficient amounts of single stranded RNAs that willform complementary base pairs with endogenous oct-3 transcripts andthereby prevent translation of oct-3 mRNA. For example, a vector can beintroduced in vivo in such a way that it is taken up by a cell andthereafter directs the transcription of an antisense RNA. Such a vectorcan remain episomal or become chromosomally integrated, as long as itcan be transcribed to produce the desired antisense RNA.

Vectors encoding an oct-3 antisense sequence can be constructed byrecombinant DNA technology methods that are standard practice in theart. Suitable vectors include plasmid vectors, viral vectors, or othertypes of vectors known or newly discovered in the art. The criterion foruse is only that the vector be capable of replicating and expressing theoct-3 antisense molecule in mammalian cells. Expression of the sequenceencoding the antisense RNA can be directed by any promoter known in theart to act in mammalian, and preferably in human, cells. Such promoterscan be inducible or constitutively active and include, but are notlimited to: the SV40 early promoter region (Bernoist et al., Nature290:304, 1981); the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto et al., Cell 22:787-797, 1988); the herpesthymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA78:1441, 1981); or the regulatory sequences of the metallothionein gene(Brinster et al., Nature 296:39, 1988).

VI. Ribozymes

Ribozyme molecules designed to catalytically cleave oct-3 mRNAtranscripts also can be used to prevent translation of oct-3 mRNA andexpression of OCT-3 polypeptides (see, for example, PCT Publication WO90/11364; Saraver et al., Science 247:1222, 1990). While variousribozymes that cleave mRNA at site-specific recognition sequences can beused to destroy oct-3 mRNAs, the use of hammerhead ribozymes ispreferred. Hammerhead ribozymes cleave mRNAs at locations dictated byflanking regions that form complementary base pairs with the targetmRNA. The sole requirement is that the target mRNA have the followingsequence of two bases: 5′-UG-3′. The construction and production ofhammerhead ribozymes is well known in the art (Haseloff et al., Nature334:585, 1988). There are numerous examples of potential hammerheadribozyme cleavage sites within the nucleotide sequence of human oct-3cDNA. Preferably, the ribozyme is engineered so that the cleavagerecognition site is located near the 5′ end of the oct-3 mRNA, i.e., toincrease efficiency and minimize the intracellular accumulation ofnon-functional mRNA transcripts.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”), such as the onethat occurs naturally in Tetrahymena Thermophila (known as the IVS orL-19 IVS RNA), and which has been extensively described by Cech and hiscollaborators (Zaug et al., Science 224:574, 1984; Zaug et al., Science231:470, 1986; Zug et al., Nature 324:429, 1986; PCT Application No. WO88/04300; and Been et al., Cell 47:207, 1986). The Cech-type ribozymeshave an eight base-pair sequence that hybridizes to a target RNAsequence, whereafter cleavage of the target RNA takes place. Theinvention encompasses those Cech-type ribozymes that target eightbase-pair active site sequences present in oct-3.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g., for improved stability, targeting, etc.), andshould be delivered to cells which express the oct-3 in vivo. Apreferred method of delivery involves using a DNA construct “encoding”the ribozyme under the control of a strong constitutive pol III or polII promoter, so that transfected cells will produce sufficientquantities of the ribozyme to destroy endogenous oct-3 messages andinhibit translation. Because ribozymes, unlike antisense molecules, arecatalytic, a lower intracellular concentration is required forefficiency.

VII. Peptide Nucleic Acids

Oct-3 nucleic acid molecules can be modified at the base moiety, sugarmoiety, or phosphate backbone to improve, for example, the stability orsolubility of the molecule or its ability to hybridize with othernucleic acid molecules. For example, the deoxyribose phosphate backboneof the nucleic acid can be modified to generate peptide nucleic acids(see Hyrup et al., Bioorganic Med. Chem. 4:5-23 (1996). As used herein,the terms “peptide nucleic acids” or “PNAs” refer to nucleic acidmimics, for example, DNA mimics, in which the deoxyribose phosphatebackbone is replaced by a pseudopeptide backbone and only the fournatural nucleobases are retained. The neutral backbone of PNAs has beenshown to allow for specific hybridization to DNA and RNA underconditions of low ionic strength. The synthesis of PNA oligomers can beperformed using standard solid phase peptide synthesis protocols asdescribed in Hyrup et al., supra; Perry-O'Keefe et al. Proc. Natl. Acad.Sci. USA 93:14670-14675 (1996).

PNAs of oct-3 can be used in therapeutic and diagnostic applications.For example, PNAs can be used as antisense or antigene agents forsequence-specific modulation of gene expression by, for example,inducing transcription or translation arrest or inhibiting replication.PNAs of oct-3 can also be used, for example, in the analysis of singlebase pair mutations in a gene by, for example, PNA-directed PCRclamping; as artificial restriction enzymes when used in combinationwith other enzymes, for example, S1 nucleases (Hyrup et al., supra); oras probes or primers for DNA sequence and hybridization (Hyrup et al.,supra; Perry-O'Keefe, supra).

In other embodiments, PNAs of oct-3 can be modified, for example, toenhance their stability or cellular uptake, by attaching lipophilic orother helper groups to the PNA, by the formation of PNA-DNA chimeras, orby the use of liposomes or other techniques of drug delivery known inthe art. For example, PNA-DNA chimeras of oct-3 can be generated thatmay combine the advantageous properties of PNA and DNA. Such chimerasallow DNA recognition enzymes, for example, RNAse H and DNA polymerases,to interact with the DNA portion while the PNA portion would providehigh binding affinity and specificity. PNA-DNA chimeras can be linkedusing linkers of appropriate lengths selected in terms of base stacking,number of bonds between the nucleobases, and orientation (Hyrup, supra).The synthesis of PNA-DNA chimeras can be performed as described inHyrup, supra, and Finn et al., Nucl. Acids Res. 24:3357-3363 (1996). Forexample, a DNA chain can be synthesized on a solid support usingstandard phosphoramidite coupling chemistry and modified nucleosideanalogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidinephosphoramidite, can be used between the PNA and the 5′ end of DNA (Maget al., Nucl. Acids Res. 17:5973-5988, 1989). PNA monomers are thencoupled in a stepwise manner to produce a chimeric molecule with a 5′PNA segment and a 3′ DNA segment (Finn et al., supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser et al., Bioorganic Med. Chem. Lett. 5:1119-11124(1975).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., Proc. Natl. Acad. Sci. USA 86:6553-6556 (1989);Lemaitre et al., Proc. Natl. Acad. Sci. USA 84:648-652 (1987); PCTPublication No. WO 88/09810, published Dec. 15, 1988) or the blood-brainbarrier (see, e.g., PCT Publication No. WO 89/10134, published Apr. 25,1988). In addition, oligonucleotides can be modified withhybridization-triggered cleavage agents (see, e.g., Krol et al.,BioTech. 6:958-976 (1988)) or integrating agents (see, e.g., Zon, Pharm.Res. 5:539-549 (1988)). To this end, the oligonucleotide may beconjugated to another molecule, for example, a peptide, hybridizationtriggered cross-linking agent, transport agent, hybridization-triggeredcleavage agent etc.

VIII. Proteins that Associate with OCT-3

The invention also features methods for identifying polypeptides thatcan associate with OCT-3, as well as the isolated interactor, forexample, proteins that alter the ability of OCT-3 to transport moleculesacross the plasma membranes of cells in which it is expressed. Anymethod that is suitable for detecting protein-protein interactions canbe employed to detect polypeptides that associate with OCT-3, whetherthese polypeptides associate with the transmembrane, intracellular, orextracellular domains of OCT-3. Among the traditional methods that canbe employed are co-immuno-precipitation, crosslinking, andco-purification through gradients or chromatographic columns of celllysates or proteins obtained from cell lysates and the use of OCT-3 toidentify proteins in the lysate that interact with OCT-3. For theseassays, the OCT-3 polypeptide can be a full length OCT-3, anextracellular domain of OCT-3, or some other suitable OCT-3 polypeptide.Once isolated, such an interacting protein can be identified and clonedand then used, in conjunction with standard techniques, to alter theactivity of the OCT-3 polypeptide with which it interacts. For example,at least a portion of the amino acid sequence of a protein thatinteracts with OCT-3 can be ascertained using techniques well known tothose of skill in the art, such as via the Edman degradation technique.The amino acid sequence obtained can be used as a guide for thegeneration of oligonucleotide mixtures that can be used to screen forgene sequences encoding the interacting protein. Screening can beaccomplished, for example, by standard hybridization or PCR techniques.Techniques for the generation of oligonucleotide mixtures and thescreening are well-known (Ausubel, supra; and “PCR Protocols: A Guide toMethods and Applications,” Innis et al., eds. Academic Press, Inc.,N.Y., 1990).

Additionally, methods can be employed that result directly in theidentification of genes that encode proteins that interact with OCT-3.These methods include, for example, screening expression libraries, in amanner similar to the well known technique of antibody probing of λgt11libraries, using labeled OCT-3 polypeptide or an OCT-3 fusion protein,for example, an OCT-3 polypeptide or domain fused to a marker such as anenzyme, fluorescent dye, a luminescent protein, or to an IgFc domain.

There are also methods available that can detect protein-proteininteraction in vivo. A method which detects protein interactions in vivois the two-hybrid system (Chien et al., Proc. Natl. Acad. Sci. USA88:9578, 1991). A kit for practicing this method is available fromClontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one plasmid includes a nucleotide sequence encodingthe DNA-binding domain of a transcription activator protein fused to anucleotide sequence encoding OCT-3, an OCT-3 polypeptide, or an OCT-3fusion protein, and the other plasmid includes a nucleotide sequenceencoding the transcription activator protein's activation domain fusedto a cDNA encoding an unknown protein which has been recombined intothis plasmid as part of a cDNA library. The DNA-binding domain fusionplasmid and the cDNA library are transformed into a strain of the yeastSaccharomyces cerevisiae that contains a reporter gene (e.g., HBS orLacZ) whose regulatory region contains the transcription activator'sbinding site. Either hybrid protein alone cannot activate transcriptionof the reporter gene: the DNA-binding domain hybrid cannot because itdoes not provide activation function, and the activation domain hybridcannot because it cannot localize to the activator's binding sites.Interaction of the two hybrid proteins reconstitutes the functionalactivator protein and results in expression of the reporter gene, whichis detected by an assay for the reporter gene product.

The two-hybrid system or related methodology can be used to screenactivation domain libraries for proteins that interact with the “bait”gene product. By way of example, and not by way of limitation, OCT-3 maybe used as the bait gene product. Total genomic or cDNA sequences arefused to the DNA encoding an activation domain. This library and aplasmid encoding a hybrid of bait OCT-3 gene product fused to theDNA-binding domain are cotransformed into a yeast reporter strain, andthe resulting transformants are screened for those that express thereporter gene. For example, a bait oct-3 gene sequence, such as oct-3 ora domain of oct-3 can be cloned into a vector such that it istranslationally fused to the DNA encoding the DNA-binding domain of theGAL4 protein. These colonies are purified and the library plasmidsresponsible for reporter gene expression are isolated. DNA sequencing isthen used to identify the proteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact withbait oct-3 gene product are to be detected can be made using methodsroutinely practiced in the art. According to the particular systemdescribed herein, for example, the cDNA fragments can be inserted into avector such that they are translationally fused to the transcriptionalactivation domain of GAL4. This library can be co-transformed along withthe bait oct-3 gene-GAL4 fusion plasmid into a yeast strain whichcontains a lacZ gene driven by a promoter which contains GAL4 activationsequence. A cDNA encoded protein, fused to GAL4 transcriptionalactivation domain, that interacts with bait oct-3 gene product willreconstitute an active GAL4 protein and thereby drive expression of theHIS3 gene. Colonies that express HIS3 can then be purified from thesestrains and used to produce and isolate the bait oct-3 gene-interactingprotein using techniques routinely practiced in the art.

IX. Detection of Oct-3 Protein or Nucleic Acid and Diagnostic Assays

The invention encompasses methods for detecting the presence of Oct-3protein or nucleic acid in a biological sample as well as methods formeasuring the level of Oct-3 protein or nucleic acid in a biologicalsample. Such methods are useful for diagnosis of disorders associatedwith aberrant expression of OCT-3.

An exemplary method for detecting the presence or absence of OCT-3 in abiological sample involves obtaining a biological sample from a testsubject and contacting the biological sample with a compound or an agentcapable of detecting an OCT-3 polypeptide or an oct-3 nucleic acid(e.g., mRNA or genomic DNA) that encodes an OCT-3 polypeptide. Apreferred agent for detecting oct-3 mRNA or genomic DNA is a labelednucleic acid probe capable of hybridizing to oct-3 mRNA or genomic DNA.The nucleic acid probe can be, for example, a full-length oct-3 nucleicacid molecule, such as a nucleic acid molecule having the sequence ofSEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:5, or a portion thereof, such asan oligonucleotide of at least 15, 30, 50, 100, 250, or 500 nucleotidesin length and sufficient to specifically hybridize under stringentconditions to oct-3 mRNA or genomic DNA.

A preferred agent for detecting an OCT-3 polypeptide is an antibodycapable of binding to an OCT-3 polypeptide, preferably an antibody witha detectable label. Antibodies can be polyclonal, or more preferably,monoclonal. An intact antibody, or a fragment thereof (e.g., Fab orF(ab′)₂) can be used. The term “labeled,” with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected with Fluorescentlylabeled strentavidin. The term “biological sample” is intended toinclude tissues, cells, and biological fluids isolated from a subject,as well as tissues, cells and fluids present within a subject. That is,the detection method of the invention can be used to detect oct-3 mRNA,an OCT-3 polypeptide, or oct-3 genomic DNA in a biological sample invitro as well as in vivo. For example, in vitro techniques for detectionof oct-3 mRNA include Northern hybridizations and in situhjybridizations. In vitro techniques for detection of an OCT-3polypeptide include enzyme linked immunosorbent assays (ELISAs), Westernblots, immunoprecioitations and immunofluorescence. In vitro techniquesfor detection of oct-3 genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of an OCT-3 polypeptideinclude introducing into a subject a labeled anti-OCT-3 antibody. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from thesubject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting an OCT-3 polypeptide,oct-3 mRNA, or oct-3 genomic DNA, such that the presence of an OCT-3polypeptide, oct-3 mRNA, or oct-3 genomic DNA is detected in thebiological sample, and comparing the presence of OCT-3 polypeptide,oct-3 mRNA, or genomic DNA in the control sample with the presence ofOCT-3 polypeptides, mRNA or genomic DNA in a test sample.

The invention also encompasses kits for detecting the presence of oct-3nucleic acid molecules or OCT-3 polypeptides in a biological sample. Forexample, the kit can contain a labeled compound or agent capable ofdetecting an OCT-3 polypeptide or an oct-3 mRNA molecule in a biologicalsample; means for determining the amount of OCT-3 in the sample; andmeans for comparing the amount of OCT-3 in the sample with a standard.The compound or agent can be packaged in a suitable container. The kitcan further contain instructions for using the kit to detect an OCT-3polypeptide or oct-3 nucleic acid molecule.

X. Prognostic Assays

The invention also encompasses prognostic assays that can be used toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant oct-3 expression or OCT-3 activity, e.g., aneurological disorder. Thus, the present invention provides a method foridentifying a disease or disorder associated with aberrant oct-3expression or OCT-3 activity in which a test sample is obtained from asubject and oct-3 nucleic acid molecules or OCT-3 polypeptides aredetected, wherein the presence of oct-3 nucleic acid or OCT-3polypeptides can be diagnostic for a subject having or at risk ofdeveloping an OCT-3 mediated disease or disorder based on the level ofoct-3 expressed or the allelic form of OCT-3 expressed. As used herein,a “test sample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,serum), a cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, polypeptide, nucleic acid, smallmolecule or other drug candidate) to treat a disease or disorderassociated with aberrant oct-3 expression or OCT-3 activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent that modulates OCT-3 expression and/oractivity. Thus, the present invention provides methods for determiningwhether a subject can be effectively treated with an agent for adisorder associated with aberrant oct-3 expression or OCT-3 activity inwhich a test sample is obtained and oct-3 nucleic acids or OCT-3polypeptides are detected (e.g., wherein the presence of a particularlevel of oct-3 expression or a particular OCT-3 allelic variant isdiagnostic for a subject that can be administered an agent to treat adisorder associated with aberrant oct-3 expression or OCT-3 activity).

The methods of the invention can also be used to detect geneticalterations in an oct-3 gene, thereby determining if a subject with thelessened gene is at risk for a disorder characterized by aberrantextracellular concentrations of molecules normally transported acrossthe cellular membrane by OCT-3. In preferred embodiments, the methodsinclude detecting, in a sample of cells from the subject, the presenceor absence of a genetic alteration characterized by at least onealteration affecting the integrity of the gene encoding an OCT-3polypeptide or the misexpression of the oct-3 gene. For example, suchgenetic alterations can be detected by ascertaining the existence of atleast one of: (1) a deletion of one or more nucleotides from an oct-3gene; (2) an addition of one or more nucleotides to an oct-3 gene; (3) asubstitution of one or more nucleotides of an oct-3 gene; (4) achromosomal rearrangement of an oct-3 gene; (5) an alteration in thelevel of a messenger RNA transcript of an oct-3 gene; (6) aberrantmodification of an oct-3 gene, such as of the methylation pattern of thegenomic DNA, (7) the presence of a non-wild type splicing pattern of amessenger RNA transcript of an oct-3 gene; and (10) inappropriatepost-translational modification of an OCT-3 polypeptide. As describedherein, there are a large number of assay techniques known in the artwhich can be used for detecting alterations in an oct-3 gene. Apreferred biological sample is a peripheral blood leukocyte sampleisolated by conventional means from a subject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR; see, e.g., U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, oralternatively, in a ligation chain reaction (LCR; see, e.g., Landegranet al., Science 241:1077-1080, 1988); and Nakazawa et al. Proc. Natl.Acad. Sci. USA 91:360-364, 1994), the latter of which can beparticularly useful for detecting point mutations in the oct-3 gene (seeAbavaya et al., Nucl. Acids Res. 23:675-681, 1995). This method caninclude the steps of collecting a sample of cells from a patient,solating nucleic acid (e.g., genomic DNA, mRNA, or both) from the cellsof the sample, contacting the nucleic acid samole with one or moreprimers which specifically hybridize to an oct-3 gene under conditionssuch that hybridization and amplification of the oct-3 nucleic acid (ifpresent) occurs, and detecting the presence or absence of anamplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli et al., Proc. Natl. Acad. Sci USA 87:1874-1878,1990), transcriptional amplification system (Kwoh et al., Proc. Natl.Acad. Sci USA 86:1173-1177, 1989), Q-Beta Replicase (Lizardi et al.,Bio/Technology 6:1197, 1988), or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of ordinary skill in the art. Thesedetection schemes are especially useful for the detection of nucleicacid molecules if such molecules are present in very low number.

In an alternative embodiment, alterations in an oct-3 gene from a samplecell can be identified by identifying changes in a restriction enzymecleavage pattern. For example, sample and control DNA is isolated,amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, e.g., U.S. Pat.No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

In other embodiments, alterations in oct-3 can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing tens to thousands of oligonucleotideprobes (Cronin et al., Human Mutation 7:244-255 (1996); Kozal et al.,Nature Medicine 2:753-759 (1996)). For example, alterations in oct-3 canbe identified in two dimensional arrays containing light-generated DNAprobes as described in Cronin et al., supra. briefly, a firsthybridization array of probes can be used to scan through long stretchesof DNA in a sample and control to identify base changes between thesequences by making linear arrays of sequential overlapping probes. Thisstep allows the identification of point mutations. This step is followedby a second hybridization array that allows the characterization ofspecific mutations by using smaller, specialized probe arrayscomplementary to all variants or mutations detected. Each mutation arrayis composed of parallel probe sets, one complementary to the wild-typegene and the other complementary to the mutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the oct-3 gene anddetect mutations by comparing the sequence of the sample oct-3 with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxim andGilbert (Proc. Natl. Acad. Sci. USA 74:560 (1977)) or Sanger (Proc.Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of avariety of automated sequencing procedures can be utilized whenperforming the diagnostic assays (Bio/Techniques 19:448 (1995))including sequencing by mass spectrometry (see, e.g. PCT InternationalPublication No. WO 94/16101; Cohen et al. Adv. Chromatogr. 36:127-162(1996); and Griffin et al. Appl. Biochem. Biotechnol. 38:147-159(1993)).

Other methods of detecting mutations in the oct-3 gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. Science230:1242 (1985)). In general, the art technique of “mismatch cleavage”starts by providing heteroduplexes formed by hybridizing (labeled) RNAor DNA containing the wild-type oct-3 sequence with potentially mutantRNA or DNA obtained from a tissue sample. The double-stranded duplexesare treated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with Si nuclease toenzymatically digest the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al., Proc.Natl. Acad. Sci. USA 85:4397 (1988); Saleeba et al., Methods Enzymol.217:286-295 (1992). In a preferred embodiment, the control DNA or RNAcan be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in oct-3 cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches (Hsu et al. Carcinogenesis 15:1657-1662 (1994)).According to an exemplary embodiment, a probe based on an oct-3 sequenceis hybridized to a cDNA or other DNA product from a test cell or cells.The duplex is treated with a DNA mismatch repair enzyme, and thecleavage products, if any, can be detected from electrophoresisprotocols or the like. See, for example, U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility can beused to identify mutations in oct-3 genes. For example, single strandconformation polymorphism (SSCP) can be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al., Proc. Natl. Acad. Sci. USA 86:2766, see also Cotton MutatRes. 285:125-144 (1993); and Hayashi Genet. Anal. Tech. Appl. 9:73-79(1992)). Single-stranded DNA fragments of sample and control oct-3nucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the method utilizes heteroduplex analysis to separate doublestranded heteroduplex molecules on the basis of changes inelectrophoretic mobility (Kee et al. Trends Genet. 7:5 (1991)).

In yet another embodiment, the movement of mutant or wild-type fragmentsin a polyacrylamide gel containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE; Myers et al. Nature313:495 (1985)). When DGGE is used as the method of analysis, DNA willbe modified to insure that it does not completely denture, for exampleby adding a GC clamp of approximately 40 bp of high-melting GC-rich DNAby PCR. In a further embodiment, a temperature gradient is used in placeof a denaturing gradient to identify differences in the mobility ofcontrol and sample DNA (Rosenbaum et al. Biophys. Chem. 265:12753(1987)).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. Nature 324;163 (1986); Saiki et al., Proc. NAtl. Acad. Sci. USA86:6230 (1989)). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonuclectides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule, sothat amplification depends on differential hybridization (Gibbs et al.,Nucl. Acids Res. 17:2437-2448 (1989)) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner, Tib/Tech 11:238 (1993)). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al., Mol. Cell Probes 6:1 (1992)). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany, Proc. Natl. Acad. Sci. USA 88:89 (1991)). in suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence of absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,for example, in a clinical setting to diagnose patient exhibitingsymptoms or a family history of a disease or disorder involving abnormalOCT-3 activity.

XI. Pharmacogenetics

Agents or modulators which have a stimulatory or inhibitory effect onOCT-3 activity (including those that alter activity by altering oct-3gene expression), identified by a screening assay described herein, canbe administered to individuals to treat, prophylactically ortherapeutically, disorders (e.g., neurological disorders) associatedwith aberrant OCT-3 activity. In conjunction with such treatment, thepharmacogenetics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) of the individual may be considered. Thus, thepharmacogenetics of the individual permits the selection of effectiveagents (e.g., drugs) for prophylactic or therapeutic treatments based ona consideration of the individual's genotype. Such pharmacogenetics canfurther be used to determine appropriate dosages and therapeuticregimens. Accordingly, the activity of OCT-3 polypeptides, expression ofoct-3 nucleic acids, or mutation content of oct-3 genes in an individualcan be determined to thereby select appropriate agents for therapeuticor prophylactic treatment of the individual.

Pharmacogenetics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, Clin. Exp. Pharmacol.Physiol. 23:983-985 (1996) and Linder, Clin. Chem. 43:254-266 (1997). Ingeneral, two types of pharmacogenetic conditions can be differentiated.Genetic conditions transmitted as a single factor altering the way drugsact on the body (altered drug action) or genetic conditions transmittedas single factors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raredefects or as polymorphisms. For example, glucose-6-phosphatedehydrogenase deficiency (G6PD) is a common inherited enzymopathy inwhich the main clinicl complication is haemolysis after ingestion ofoxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans)and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g.

N-acetyltransferase (NAT2) and cytochrome P450 enzymes CYP2D6 andCYP2C19) has provided an explanation as to why some patients do notobtain the expected drug effects or show exaggerated drug response andserious toxicity after taking the standard and safe dose of a drug.These polymorphisms are expressed in two phenotypes in the population,the extensize metabolizer (EM) and poor metabolizer (PM). The prevalenceof PM is different among different populations. For example, the genecoding for CYP2D6 is highly polymorphic and several mutations have beenidentified in PM, which all lead to the absence of functional CYP2D6.Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experienceexaggerated drug response and side effects when they receive standarddoses. If a metabolite is the active therapeutic moiety, PM show notherapeutic response, as demonstrated for the analgesic effect ofcodeine mediated by its CYP2D6-formed metabolite morphine. The otherextreme is the so called ultra-rapid metabolizers who do not respond tostandard doses. Recently, the molecular basis of ultra-rapid metabolismhas been identified to be due to CYP2D6 gene amplification.

Thus, the activity of OCT-3 polypeptide, expression of oct-3 nucleicacid, or mutation content of oct-3 gene in an individual can bedetermined to thereby select appropriate agents for therapeutic orprophylactic treatment of the individual. In addition, pharmacogeneticstudies can be used to apply genotyping of polymorphic alleles encodingdrug-metabolizing enzymes to the identification of an individual's drugresponsiveness phenotype. This knowledge, when applied to dosing or drugselection, can avoid adverse reactions or therapeutic failure and thusenhance therapeutic or prophylactic efficiency when treating a subjectwith an OCT-3 modulator, such as a modulator identified by one of theexemplary screening assays described herein.

XII. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs, compounds) on theexpression of oct-3 or the activity OCT-3 (e.g., the ability to modulatethe symptoms associated with neurological disorders) can be applied notonly in basic drug screening, but also in clinical trials. For example,the effectiveness of an agent determined by a screening assay asdescribed herein to increase oct-3 gene expression, OCT-3 polypeptidelevels, or upregulate OCT-3 activity, can be monitored in clinicaltrials of subjects exhibiting decreased oct-3 gene expression, decreasedOCT-3 polypeptide levels, or downregulated OCT-3 activity.Alternatively, the effectiveness of an agent determined by a screeningassay to decrease oct-3 gene expression, decrease OCT-3 polypeptidelevels, or downregulate OCT-3 activity, can be monitored in clinicaltrials of subjects exhibiting increased oct-3 gene expression, increasedOCT-3 polypeptide levels, or upregulated OCT-3 activity. In suchclinical trials, the expression of oct-3 or activity of OCT-3 can beused as a “read out” or marker of the responsiveness of a particularcell.

For example, and not by way of limitation, genes, including oct-3, thatare modulated in cells by treatment with an agent (e.g., a compound,drug, or small molecule) that modulates OCT-3 activity (e.g., identifiedin a screening assay as described herein) can be identified. Thus, tostudy the effect of agents on neurolgoical disorders, for example, in aclinical trial, cells can be isolated and RNA prepared and analyzed forthe level of oct-3 expression and other genes implicated in theneurological disorder. The levels of gene expression (i.e., a geneexpression pattern) can be quantified by Northern blot analysis orRT-PCR, as described herein, or alternatively by measuring the amount ofpolypeptide produced, by one of the methods described herein, or bymeasuring the levels of activity of OCT-3 or other genes. In this way,the gene expression pattern can serve as an indicative marker of thephysiological response of the cells to the agent. Accordingly, thisresponse state can be determined before, and at various points during,treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, polypeptide, nucleicacid, small molecule, or other drug candidate identified by theascreening assays described herein) comprising the steps of (1)obtaining a pre-administration sample from a subject prior toadministration of the agent; (2) detecting the level of expression of anOCT-3 polypeptide or oct-3 mRNA in the pre-administration sample, or thelevel or activity of oct-3; (3) obtaining one or morepost-administration samples from the subject; (4) detecting the level ofexpression of oct-3 mRNA or the level or activity of the OCT-3polypeptide in the post-administration sample; (5) comparing the levelof expression of oct-3 mRNA in the pre-administration sample with thatin the post-administration sample, or comparing the level or activity ofthe OCT-3 polypeptide in the pre-administration sample with that in thepost-administration sample; and (6) altering the administration of theagent to the subject accordingly.

XIII. Screening Assays for Compounds that Modulate OCT-3 Expression orActivity

The invention also encompasses methods for identifying compounds thatinteract with OCT-3 (or a domain of OCT-3) including, but not limitedto, compounds that interfere with the interaction of OCT-3 withtransmembrane or intracellular proteins which regulate OCT-3 activityand compounds which modulate OCT-3 activity. Also encompasses are methodfor identifying compounds which bind to Oct-3 gene regulatory sequences(e.g., promoter sequences) and which may modulate Oct-3 gene expression.

The compounds which may be screened in accordance with the inventioninclude, but are not limited to peptides, antibodies and fragmentsthereof, and other organic compounds that bind to OCT-3 and increase ordecrease activity.

Such compounds may include, but are not limited to, peptides such as,for example, soluble peptldes, including but not limited to members ofrandom peptide libraries; (Lam et al., Nature 354:82-84, 1991; Houghtenet al., Nature 354:84-86, 1991), and combinatorial chemistry-derivedmolecular library made of D- and/or L- configuration amino acids,phosphopeptides (including, but not limited to, members of random orpartially degenerate, directed phosphopeptide libraries; Songyang, etal., Cell 72:767-778, 1993), antibodies (including, but not limited to,polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or singlechain antibodies, and FAb, F(ab′)₂ and FAb expression library fragments,and epitope-binding fragments thereof), and small organic or inorganicmolecules.

Other compounds which can be screened in accordance with the inventioninclude but are not limited to small organic molecules that are able togain entry into an appropriate cell and affect the expression of theOct-3 gene or activity of Oct-3 protein.

Computer modelling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate OCT-3 expression or activity. Having identified such a compoundor composition, the active sites or regions are identified. Such activesites might typically be a binding for a natural modulator of activity.The active site can be identified using methods known in the artincluding, for example, from the amino acid sequences of peptides, fromthe nucleotide sequences of nucleic acids, or from study of complexes ofthe relevant compound or composition with its natural ligand. In thelatter case, chemical or X-ray crystallographic methods can be used tofind the active site by finding where on the factor the modulator (orligand) is found.

Next, the three dimensional geometric structure of the active site isdetermined. This can be done by known methods, including X-raycrystallography, which can determine a complete molecular structure. Onthe other hand, solid or liquid phase NMR can be used to determinecertain intra-molecular distances. Any other experimental method ofstructure determination can be used to obtain partial or completegeometric structures. The geometric structures may be measured with acomplexed modulator (ligand), natural or artificial, which may increasethe accuracy of the active site structure determined.

If an incomplete or insufficiently accurate structure is determined, themethods of computer based numerical modelling can be used to completethe structure or improve its accuracy. Any recognized modelling methodmay be used, including parameterized models specific to particularbiopolymers such as proteins or nucleic acids, molecular dynamics modelsbased on computing molecular motions, statistical mechanics models basedon thermal ensembles, or combined models. For most types of models,standard molecular force fields, representing the forces betweenconstituent atoms and groups, are necessary, and can be selected fromforce fields known in physical chemistry. The incomplete or lessaccurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

Finally, having determined the structure of the active site, eitherexperimentally, by modeling, or by a combination, candidate modulatingcompounds can be identified by searching databases containing compoundsalong with information on their molecular structure. Such a search seekscompounds having structures that match the determined active sitestructure and that interact with the groups defining the active site.Such a seach can be manual, but is preferably computer assisted. Thesecompounds found from this search are potential OCT-3 modulatingcompounds.

Alternatively, these methods can be used to identify improved modulatingcompounds from a previously identified modulating compound or ligand.The composition of the known compound can be modified and the structuraleffects of modification can be determined using the experimental andcomputer modelling methods described above applied to the newcomposition. The altered structure is then compared to the active sitestructure of the compound to determine if an improved fit or interactionresults. In this manner systematic variations in composition, such as byvarying side groups, can be quickly evaluated to obtain modifiedmodulating compounds or ligands of improved specificity or activity.

Examples of molecular modelling systems are the CHARMm and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMm performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modelling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modelling of drugs interactive withspecific proteins, such as Rotivinen et al. Acta Pharmaceutical Fennica97:159-166, 1993; Ripka, New Scientist 54-57 (Jun. 16, 1988); McKinalyand Rossmann, Annu. Rev. Pharmacol. Toxiciol. 29:111-122, 1989; Perryand Davies, OSAR: Quantitative Structure-Activity Relationships in DrugDesign, pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 989 Proc.R. Soc. Lond. 236:125-140 and 141-162; and, with respect to a modelreceptor for nucleic acid components, Askew et al., J. Am. Chem. Soc.111:1082-1090, 1989. Other computer programs that screen and graphicallydepict chemicals are available from companies such as BioDesign, Inc.(Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario, Canada), andHypercube, Inc. (Cambridge, Ontario). Although these are primarilydesigned for application to drugs specific to particular proteins, theycan be adapted to design of drugs specific to regions of DNA or RNA,once that region is identified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichare inhibitors or activators of OCT-3 activity (ion transport).

Compounds identified via assays such as those described herein may beuseful, for example, in elaborating the biological function of OCT-3 andfor the treatment of disorders associated with aberrant OCT-3 activityor expression. Assays for testing the effectiveness of compoundsidentified with the above-described techniques are are discussed below.

In vitro systems may be designed to identify compounds capable ofinteracting with OCT-3 (or a domain of OCT-B3). Compounds identified maybe useful, for example, in modulating the activity of wild type and/ormutant OCT-3; may be useful in elaborating the biological function ofthe OCT-3; may be utilized in screens for identifying compounds thatdisrupt normal OCT-3 interactions; or may in themselves disrupt suchinteractions.

The principle of the assays used to identify compounds that bind to theOCT-3 involves preparing a reaction mixture of OCT-3 (or a doaminthereof) and the test compound under conditions and for a timesufficient to allow the two components to interact and bind, thusforming a complex which can be removed and/or detected in the reactionmixture. The OCT-3 species used can vary depending upon the goal of thescreening assay. In some situations it is preferable to employ a peptidecorresponding to a domain of OCT-3 fused to a heterologous protein orpolypeptide that affords advantages in the assay system (e.g., labeling,isolation of the resulting complex, etc.) can be utilized.

The screening assays can be conducted in a variety of ways. For example,one method to conduct such an assay involves anchoring the OCT-3protein, polypeptide, peptide or fusion protein or the test substanceonto a solid phase and detecting OCT-3/test compound complexes anchoredon the solid phase at the end of the reaction. In one embodiment of sucha method, the OCT-3 reactant may be anchored onto a solid surface, andthe test compound, which is not anchored, may be labeled, eitherdirectly or indirectly.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the previously nonimmobilizedcomponent (the antibody, in turn, may be directly labeled or indirectlylabeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; e.g., using an immobilized antibody specific for OCT-3protein, polypeptide, peptide or fusion protein or the test compound toanchor any complexes formed in solution, and a labeled antibody specificfor the other component of the possible complex to detect anchoredcomplexes.

Alternatively, cell-based assays can be used to identify compounds thatinteract with OCT-3. To this end, cell lines that express OCT-3, or celllines that have been genetically engineered to express OCT-3 can beused.

XIV. Assays for Compounds that Interfere with Proteins that Interactwith OCT-3

Proteins that interact with the OCT-3 are referred to, for purposes ofthis discussion, as “binding partners”. Such binding partners areinvolved in regulating OCT-3 activity. Therefore, it is desirable toidentify compounds that interfere with or disrupt the interaction ofsuch binding partners with OCT-3. Such compounds may be useful inregulating the activity of the OCT-3 and treating disorders associatedwith aberrant OCT-3 activity.

The basic principle of the assay systems used to identify compounds -hatinterfere with the interaction between the OCT-3 and its binding partneror partners involves preparing a reaction mixture containing OCT-3protein, polypeptide, peptide or fusion protein and the binding partnerunder conditions and for a time sufficient to allow the two to interactand bind, thus forming a complex. In order to test a compound forinhibitory activity, the reaction mixture is prepared in the presenceand absence of the test compound. The test compound may be initiallyincluded in the reaction mixture, or may be added at a time subsequentto the addition of the OCT-3 moiety and its binding partner. Controlreaction mixtures are incubated without the test compound or with anon-active control compound. The formation of any complexes between theOCT-3 moiety and the binding partner is then detected. The formation ofa complex in the control reaction, but not in the reaction mixturecontaining the test compound, indicates that the compound interfereswith the interaction of the OCT-3 and the interactive binding partner.Additionally, complex formation within reaction mixtures containing thetest compound and normal OCT-3 protein may also be compared to complexformation within reaction mixtures containing the test compound and amutant OCT-3. This comparison may be important in those cases wherein itis desirable to identify compounds that disrupt interactions of mutantbut not normal OCT-3.

The assay for compounds that interfere with the interaction of the OCT-3and binding partners can be conducted in a heterogeneous or homogeneousformat. Heterogeneous assavs involve anchoring either the OCT-3 protein,polypeptide, peptide, or fusion protein, or the binding partner onto asolid phase and detecting complexes anchored on the solid phase at theend of the reaction. In homogeneous assays, the entire reaction iscarried out in a liquid phase. In either approach, the order of additionof reactants can be varied to obtain different information about thecompounds being tested. For example, test compounds that interfere withthe interaction by competition can be identified by conducting thereaction in the presence of the test substance; i.e., by adding the testsubstance to the reaction mixture prior to or simultaneously with theOCT-3 moiety and interactive binding partner. Alternatively, testcompounds that disrupt preformed complexes, e.g., compounds with higherbinding constants that displace one of the components from the complex,can be tested by adding the test compound to the reaction mixture aftercomplexes have been formed. The various formats are described brieflybelow.

In a heterogeneous assay system, either the OCT-3 moiety or theinteractive binding partner, is anchored onto a solid surface, while thenon-anchored species is labeled, either directly or indirectly. Inpractice, microtiter plates are conveniently utilized. The anchoredspecies may be immobilized by non-covalent or covalent attachments.Non-covalent attachment may be accomplished simply by coating the solidsurface with a solution of OCT-3 (or a doamin thereof) or bindingpartner and drying. Alternatively, an immobilized antibody specific forthe species to be anchored may be used to anchor the species to thesolid surface. The surfaces may be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface, e.g., using a directly or indirectly labeled antibodyspecific for the initially non-immobilized species. Depending upon theorder of addition of reaction components, test compounds which inhibitcomplex formation or which disrupt preformed complexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected, e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds which inhibit complex or which disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the OCT-3 moiety and theinteractive binding partner is prepared in which either the OCT-3 or itsbinding partners is labeled, but the signal generated by the label isquenched due to formation of the complex (see, e.g., U.S. Pat. No.4,109,496 by Rubenstein which utilizes this approach for immunoassays).The addition of a test substance that competes with and displaces one ofthe species from the preformed complex will result in the generation ofa signal above background. In this way, test substances which disruptOCT-3/intracellular binding partner interaction can be identified.

In a particular embodiment, an OCT-3 fusion can be prepared forimmobilization. For example, the OCT-3 or a peptide fragment thereof canbe fused to a glutathione-S-transferase (GST) gene using a fusionvector, such as pGEX-5X-1, in such a manner that its binding activity ismaintained in the resulting fusion protein. The interactive bindingpartner can be purified and used to raise a monoclonal antibody, usingmethods routinely practiced in the art. This antibody can be labeledwith the radioactive isotope ¹²⁵I, for example, by methods routinelypracticed in the art. In a heterogeneous assay, the GST-OCT-3 fusionprorotein can be anchored to glutathione-agarose beads. The interactivebinding partner can then be added in the presence or absence of the testcompound in a manner that allows interaction and binding to occur. Atthe end of the reaction period, unbound material can be washed away, andthe labeled monoclonal antibody can be added to the system and allowedto bind to the complexed components. The interaction between OCT-3 andthe interactive binding partner can be detected by measuring the amountof radioactivity that remains associated with the glutathione-agarosebeads. A successful inhibition of the interaction by the test compoundwill result in a decrease in measured radioactivity.

Alternatively, the GST-OCT-3 fusion protein and the interactive bindingpartner can be mixed together in liquid in the absence of the solidglutathione-agarose beads. The test compound can be added either duringor after the species are allowed to interact. This mixture can then beadded to the glutathione-agarose beads and unbound material is washedaway. Again the extent of inhibition of the OCT-3/binding partnerinteraction can be detected by adding the labeled antibody and measuringthe radioactivity associated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the OCT-3 and/or the interactive or binding partner (in cases wherethe binding partner is a protein), in place of one or both of the fulllength proteins. Any number of methods routinely practiced in the artcan be used to identify and isolate the binding sites. These methodsinclude, but are not limited to, mutagenesis of the gene encoding one ofthe proteins and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can then be selected.Sequence analysis of the genes encoding the respective proteins willreveal the mutations that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain may remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for theintracellular binding partner is obtained, short gene segments can beengineered to express peptide fragments of the protein, which can thenbe tested for binding activity and purified or synthesized.

XV. Methods for Reducing Oct-3 Expression

Oct-3 expression can be reduced through the use of modulatory compoundsidentified through the use of the screening methods described above. Inaddition, endogenous oct-3 gene expression can also be reduced byinactivating or “knocking out” the oct-3 gene or its promoter usingtargeted homologous recombination (see, for example, U.S. Pat. No.5,464,764). For example, a mutant, non-functional oct-3 (or a completelyunrelated DNA sequence) flanked by DNA homologous to the endogenousoct-3 gene (either the coding regions or regulatory regions of the oct-3gene) can be used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express oct-3 in vivo.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the oct-3 gene. Such approaches areparticularly suited for use in developing animal models to study therole of OCT-3; in this instance, modifications to ES (embryonic stem)cells can be used to generate animal offspring with an inactive oct-3gene. However, a knock out approach can be adapted for use in humans,provided the recombinant DNA constructs are directly administered ortargeted to the required site in vivo using appropriate viral vectors.

Alternatively, endogenous oct-3 gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the oct-3 gene (i.e., the oct-3 promoter and/or enhancers) toform triple helical structures that prevent transcription of the oct-3gene in target cells in the body (Helene, Anticancer Drug Res. 6:569,1981; Helene et al., Ann. N.Y. Acad. Sci. 660:27, 1992; and Maher,Bioassays 14:807, 1992).

In addition, as discussed above, anti-sense moleceules, ribozymes, andpeptide nucleic acids can be used to reduce oct-3 expression.

XVI. Assays for the Identification of Compounds that AmeliorateDisorders Associated with Aberrant OCT-3 Expression or Activity

Compounds, including, but not limited to, compounds identified via assaytechniques such as those described above may be useful for the treatmentof disorders associated with aberrant OCT-3 activity.

While animal model-based assays are particularly useful for theidentification of such therapeutic compounds, cell-based assay systemsare also very useful, particularly in combination with animal-modelbased assays. Such cell-based systems can include, for example,recombinant or non-recombinant cells which express OCT-3. The effect ofa selected modulatory compound on OCT-3 expression can be measured usingany of the above-described techniques for measuring OCT-3 protein orOct-3 mRNA, and the effect of a selected modulatory compound on OCT-3activity can be measured by measuring the flux of a molecule transportedby OCT-3.

XVII. Effective Dose

Toxicity and therapeutic efficacy of the polypeptides of the inventionand the compounds that modulate their expression or activity can bedetermined by standard pharmaceutical procedures, using either cells inculture or experimental animals to determine the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and lt can be expressed asthe ratio LD₅₀/ED₅₀. Polypeptides or other compounds that exhibit largetherapeutic indices are preferred. While compounds that exhibit toxicside effects may be used, care should be taken to design a deliverysystem that targets such compounds to the site of affected tissue inorder to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (that is, the concentrationof the test compound which achieves a half-maximal inhibition ofsymptoms) as determined in cell culture. Such information can be used tomore accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by high performance liquid chromatography.

XVIII. Formulations and Use

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in a conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by inhalation or insufflation (eitherthrough the mouth or the nose) or oral, buccal, parenteral or rectaladministration.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone, orhydroxypropyl methylcellulose); Fillers (e.g., lactose, microcrystallinecellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc, or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated to give controlled release ofthe active compound.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, for example, gelatin for use in an inhaleror insufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, for example, by bolus injection or continuous infusion.Formulations for injection may be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions may take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, for example, sterile pyrogen-freewater, before use.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, for example, containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (e.g., subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (e.g., as an emulsion in an acceptable oil) or ion exchangeresins, or as sparingly soluble derivatives, for example, as a sparinglysoluble salt.

The compositions can, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack can, for example, comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

The therapeutic compositions of the invention can also contain a carrieror excipient, many of which are known to persons of ordinary skill inthe art. Excipients that can be used include buffers (e.g., citratebuffer, phosphate buffer, acetate buffer, and bicarbonate buffer), aminoacids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g.,serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol,and glycerol.

The nucleic acids, polypeptides, antibodies, or other modulatorycompounds of the invention (i.e., compounds that alter the expression ofoct-3 or the activity of OCT-3) can be administered by any standardroute of administration. For example, administration can be parenteral,intravenous, subcutaneous, intramuscular, intracranial, intraorbital,opthalmic, intraventricular, intracapsular, intraspinal, intracisternal,intraperitoneal, zransmucosal, or oral. The modulatory compound can beFormulated in various ways, according to the corresponding route ofadministration. For example, liquid solutions can be made for ingestionor injection; gels or powders can be made for ingestion, inhalation, ortopical application. Methods for making such formulations are well knownand can be found in, for example, “Remington's Pharmaceutical Sciences.”It is expected that the preferred route of administration will beintravenous.

XIX. Compounds Transported by OCT-3

Compounds that are transported across cellular membranes by an OCT-3polypeptide of the invention can be identified by transiently expressingan oct-3 gene of the invention in cells (e.g., 293 cells) and measuringthe transport of candidate molecules across the plasma membranes ofthose cells. The expression of an OCT-3 polypeptide could be confirmedby, for example, performing immunohistochemistry co detect either OCT-3or an epitope tag placed on the OCT-3 polypeptide. To measure thetransport of candidate molecules across the plasma membrane, one coulddirectly measure the uptake of radiolabelled molecules such as TEA,dopamine, serotonin, histamine, and acetylcholine. This assay could beperformed, for example, by incubating the cells expressing the OCT-3transporter with radiolabelled candidate molecules for 1 hour at roomtemperature, washing the cells with an ice cold buffer, lysing thecells, and measuring the radioactivity in scintillant in a beta counter.Persons of ordinary skill in the art may consult Martel et al. (Arch.Pharmacol. 354:320-326, 1996), if necessary, for a more detailedprotocol.

The assay described above can be used to determine whether any givenpolypeptide functions as an OCT-3 polypeptide of the invention. To makethis determination, one would simply perform the assay using the OCT-3polypeptide described herein and a second, putative OCT-3 polypeptide(such as a polypeptide that differs from SEQ ID NO:2 by the addition,deletion, or substitution of one or more amino acid residues), inparallel. The putative OCT-3 polypeptide would prove to be within thescope of the present invention if it exhibited 20%, 40%, or 50% of theactivity of the full-length, mature form of OCT-3. Preferably, acandidate OCT-3 molecule would exhibit 75%, 80%, or even 90% (or more,e.g., 95% or even 99%) of the activity of OCT-3, as disclosed herein.Candidate OCT-3 polypeptides that are substantially identical to SEQ IDNO:2 may even exhibit greater biological activity (i.e., serve as moreeffective transporters) than the wild-type polypeptide itself.

XX. EXAMPLES

Examples 1 and 2 describe the identification and characterization of ratand human oct-3 nucleic acid molecules, respectively.

Example 1

A cDNA library in plasmid pMET7 was prepared using oligo dT-purified RNAisolated from adult rat frontal cortex tissue. The library was plated,colonies were selected randomly, and the 5′ ends of the cDNA insertswere sequenced (5′ sample sequencing). Each sequence (5′ expressedsequence tag (EST)) was compared with sequences in public databasesusing the BLAST algorithm. One such 5′ EST had an extremely low degreeof similarity with OCT-2 sequences identified in pig and rat, and lessersimilarity to nucleic acid molecules encoding other organic cationtransport proteins. The sequence comparison indicated that the cloneIdentified from the rat library was a partial length clone having acoding sequence that extended from approximately the 5′ end of the sixthputative transmembrane domain through the 3′ end of the gene. This clonewas sequenced by sequentially “walking” from both the 5′ and the 3′ endsof the molecule. A hydropathy plot of the coding region indicated 7transmembrane domains corresponding, approximately, in cosition to thesixth through the twelfth transmembrane domains of OCT-2. No stop codonswere found in the homologous reading frame and a poly-A tail wasdetected. These features indicate that this is a legitimate, functionalmRNA, rather than a pseudogene.

The 5′ EST sequence was used to prepare a DNA probe that was labelledand hybridized to Northern blots. Hybridization to a blot containingseveral rat tissues (a rat multiple tissue Northern blot was purchasedfrom Clontech) indicated a robust signal corresponding to a 2.3 kbtranscript predominantly in brain tissues. Extended exposure of theaucoradiogram prepared from the Northern blot indicated the presence ofsimilar bands in a subset of other tissues including kidney, heart,lung, testis, spleen, and liver.

The rat RNA probe was also hybridized to a Northern blot containingsamples from different regions of the human CNS, including spinal cordand cortex. A single band was observed in all samples. The intensity oflabelling was similar in all samples, although somewhat reduced in thespinal cord.

The rat OCT-3 clone was used to make probes for in silu hybridizationexperiments. Experiments with various coronal sections of rat brainshowed strong hybridization at the cell bodies of neurons. Nosignificant hybridization was observed to non-neuronal cells. Thehybridization signal at some neurons, for example, Purkinje cells, wasrelatively weak, while in other cells, for example, granule cells of thehippocampus and cerebellum, the hybridization signal was intense.

The sequence of th e rat cDNA clone described in this example and thepredicted amino acid sequence is shown in FIGS. 3A-3B.

Example 2

The rat OCT-3 clone described in Example 1 was used to identify aportion of a human OCT-3 cDNA clone, which was used to screen a humancerebellum cDNA library. Seventy-seven clones were identified, and the5′ ends of 10 of these were sequenced and found to represent the samegene. Restriction digests of the plasmids showed two classes of insertlengths. One insert was approximately 1.5 kb and the other wasapproximately 2.4 kb. Two clones, designated “g” and “a,” were selectedas examples of each class and fully sequenced. Clone “a” has 12hydrorhobic transmebrane regions (as predicted by the Kyte and Doolittlealgorithm), which is characteristic of transporter proteins. Thesequence of clone “a” is shown in FIGS. 1A-1C (SEQ ID NO:1) Clone “g”represen ts a truncated mRNA whose coding region includes the first sixTM domains. The sequence of clone “g” is shown in FIGS. 2A-2B (SEQ IDNO:3).

XXI. Deposit Statement

The subject clones have been deposited under conditions that assure thataccess to them will be available during the pendency of the patentapplication in which they are disclosed to those determined by theCommissioner of Patents and Trademarks to be entitled thereto under 37C.F.R. 1.14 and 35 U.S.C. § 122. More specifically, the clones describedherein as “g” and “a” were deposited with the American Type CultureCollection and assigned accession numbers 98518 and 98519, respectively.

17 2460 base pairs nucleic acid single linear Genomic DNA not providedCoding Sequence 498...2057 1 GTCGACCCAC GCGTCCGCCC ACGCGTCCGG GAGGCGCCCGCGGCTGCAGA GCTGCAGAGC 60 GGGATCTCTT CGAGCTGTCT GTGTCCGGGC AGCCGGCGCGCAACTGAGCC AGAGGACAGC 120 GCATCCTTTC GGCGCGGGCC GGCAGGGCCC CTGCGGTCGGCAAGCTGGCT CCCCGGGTGG 180 CCACCGGGAC CCCCGAGCCC AATGGCGGGG GCGGCGGCAAAATCGACAAC ACTGTAGAGA 240 TCACCCCCAC CTCCAACGGA CAGGTCGGGA CCCTCGGAGATGCGGTGCCC ACGGAGCAGC 300 TGCAGGGTGA GCGGGAGCGC GAGCGGGAGG GGGAGGGAGACGCGGGCGGC GACGGACTGG 360 GCAGCAGCCT GTCGCTGGCC GTGCCCCCAG GCCCCCTCAGCTTTGAGGCG CTGCTCGCCC 420 AGGTGGGGGC GCTGGGCGGC GGCCAGCAGC TGCAGCTCGGCCTCTGCTGC CTGCCGGTGC 480 TCTTCGTGGC TCTGGGC ATG GCC TCG GAC CCC ATC TTCACG CTG GCG CCC 530 Met Ala Ser Asp Pro Ile Phe Thr Leu Ala Pro 1 5 10CCG CTG CAT TGC CAC TAC GGG GCC TTC CCC CCT AAT GCC TCT GGC TGG 578 ProLeu His Cys His Tyr Gly Ala Phe Pro Pro Asn Ala Ser Gly Trp 15 20 25 GAGCAG CCT CCC AAT GCC AGC GGC GTC AGC GTC GCC AGC GCT GCC CTA 626 Glu GlnPro Pro Asn Ala Ser Gly Val Ser Val Ala Ser Ala Ala Leu 30 35 40 GCA GCCAGC GCC GCC AGC CGT GTC GCC ACC AGT ACC GAC CCC TCG TGC 674 Ala Ala SerAla Ala Ser Arg Val Ala Thr Ser Thr Asp Pro Ser Cys 45 50 55 AGC GGC TTCGCC CCG CCG GAC TTC AAC CAT TGC CTC AAG GAT TGG GAC 722 Ser Gly Phe AlaPro Pro Asp Phe Asn His Cys Leu Lys Asp Trp Asp 60 65 70 75 TAT AAT GGCCTT CCT GTG CTC ACC ACC AAC GCC ATC GGC CAG TGG GAT 770 Tyr Asn Gly LeuPro Val Leu Thr Thr Asn Ala Ile Gly Gln Trp Asp 80 85 90 CTG GTG TGT GACCTG GGC TGG CAG GTG ATC CTG GAG CAG ATC CTC TTC 818 Leu Val Cys Asp LeuGly Trp Gln Val Ile Leu Glu Gln Ile Leu Phe 95 100 105 ATC TTG GGC TTTGCC TCC GGC TAC CTG TTC CTG GGT TAC CCC GCA GAC 866 Ile Leu Gly Phe AlaSer Gly Tyr Leu Phe Leu Gly Tyr Pro Ala Asp 110 115 120 AGA TTT GGC CGTCGC GGG ATT GTG CTG CTG ACC TTG GGG CTG GTG GGC 914 Arg Phe Gly Arg ArgGly Ile Val Leu Leu Thr Leu Gly Leu Val Gly 125 130 135 CCC TGT GGA GTAGGA GGG GCT GCT GCA GGC TCC TCC ACA GGC GTC ATG 962 Pro Cys Gly Val GlyGly Ala Ala Ala Gly Ser Ser Thr Gly Val Met 140 145 150 155 GCC CTC CGATTC CTC TTG GGC TTT CTG CTT GCC GGT GTT GAC CTG GGT 1010 Ala Leu Arg PheLeu Leu Gly Phe Leu Leu Ala Gly Val Asp Leu Gly 160 165 170 GTC TAC CTGATG CGC CTG GAG CTG TGC GAC CCA ACC CAG AGG CTT CGG 1058 Val Tyr Leu MetArg Leu Glu Leu Cys Asp Pro Thr Gln Arg Leu Arg 175 180 185 GTG GCC CTGGCA GGG GAG TTG GTG GGG GTG GGA GGG CAC TTC CTG TTC 1106 Val Ala Leu AlaGly Glu Leu Val Gly Val Gly Gly His Phe Leu Phe 190 195 200 CTG GGC CTGGCC CTT GTC TCT AAG GAT TGG CGA TTC CTA CAG CGA ATG 1154 Leu Gly Leu AlaLeu Val Ser Lys Asp Trp Arg Phe Leu Gln Arg Met 205 210 215 ATC ACC GCTCCC TGC ATC CTC TTC CTG TTT TAT GGC TGG CCT GGT TTG 1202 Ile Thr Ala ProCys Ile Leu Phe Leu Phe Tyr Gly Trp Pro Gly Leu 220 225 230 235 TTC CTGGAG TCC GCA CGG TGG CTG ATA GTG AAG CGG CAG ATT GAG GAG 1250 Phe Leu GluSer Ala Arg Trp Leu Ile Val Lys Arg Gln Ile Glu Glu 240 245 250 GCT CAGTCT GTG CTG AGG ATC CTG GCT GAG CGA AAC CGG CCC CAT GGG 1298 Ala Gln SerVal Leu Arg Ile Leu Ala Glu Arg Asn Arg Pro His Gly 255 260 265 CAG ATGCTG GGG GAG GAG GCC CAG GAG GCC CTG CAG GAC CTG GAG AAT 1346 Gln Met LeuGly Glu Glu Ala Gln Glu Ala Leu Gln Asp Leu Glu Asn 270 275 280 ACC TGCCCT CTC CCT GCA ACA TCC TCC TTT TCC TTT GCT TCC CTC CTC 1394 Thr Cys ProLeu Pro Ala Thr Ser Ser Phe Ser Phe Ala Ser Leu Leu 285 290 295 AAC TACCGC AAC ATC TGG AAA AAT CTG CTT ATC CTG GGC TTC ACC AAC 1442 Asn Tyr ArgAsn Ile Trp Lys Asn Leu Leu Ile Leu Gly Phe Thr Asn 300 305 310 315 TTCATT GCC CAT GCC ATT CGC CAC TGC TAC CAG CCT GTG GGA GGA GGA 1490 Phe IleAla His Ala Ile Arg His Cys Tyr Gln Pro Val Gly Gly Gly 320 325 330 GGGAGC CCA TCG GAC TTC TAC CTG TGC TCT CTG CTG GCC AGC GGC ACC 1538 Gly SerPro Ser Asp Phe Tyr Leu Cys Ser Leu Leu Ala Ser Gly Thr 335 340 345 GCAGCC CTG GCC TGT GTC TTC CTG GGG GTC ACC GTG GAC CGA TTT GGC 1586 Ala AlaLeu Ala Cys Val Phe Leu Gly Val Thr Val Asp Arg Phe Gly 350 355 360 CGCCGG GGC ATC CTT CTT CTC TCC ATG ACC CTT ACC GGC ATT GCT TCC 1634 Arg ArgGly Ile Leu Leu Leu Ser Met Thr Leu Thr Gly Ile Ala Ser 365 370 375 CTGGTC CTG CTG GGC CTG TGG GAT TAT CTG AAC GAG GCT GCC ATC ACC 1682 Leu ValLeu Leu Gly Leu Trp Asp Tyr Leu Asn Glu Ala Ala Ile Thr 380 385 390 395ACT TTC TCT GTC CTT GGG CTC TTC TCC TCC CAA GCT GCC GCC ATC CTC 1730 ThrPhe Ser Val Leu Gly Leu Phe Ser Ser Gln Ala Ala Ala Ile Leu 400 405 410AGC ACC CTC CTT GCT GCT GAG GTC ATC CCC ACC ACT GTC CGG GGC CGT 1778 SerThr Leu Leu Ala Ala Glu Val Ile Pro Thr Thr Val Arg Gly Arg 415 420 425GGC CTG GGC CTG ATC ATG GCT CTA GGG GCG CTT GGA GGA CTG AGC GGC 1826 GlyLeu Gly Leu Ile Met Ala Leu Gly Ala Leu Gly Gly Leu Ser Gly 430 435 440CCG GCC CAG CGC CTC CAC ATG GGC CAT GGA GCC TTC CTG CAG CAC GTG 1874 ProAla Gln Arg Leu His Met Gly His Gly Ala Phe Leu Gln His Val 445 450 455GTG CTG GCG GCC TGC GCC CTC CTC TGC ATT CTC AGC ATT ATG CTG CTG 1922 ValLeu Ala Ala Cys Ala Leu Leu Cys Ile Leu Ser Ile Met Leu Leu 460 465 470475 CCG GAG ACC AAG CGC AAG CTC CTG CCC GAG GTG CTC CGG GAC GGG GAG 1970Pro Glu Thr Lys Arg Lys Leu Leu Pro Glu Val Leu Arg Asp Gly Glu 480 485490 CTG TGT CGC CGG CCT TCC CTG CTG CGG CAG CCA CCC CCT ACC CGC TGT 2018Leu Cys Arg Arg Pro Ser Leu Leu Arg Gln Pro Pro Pro Thr Arg Cys 495 500505 GAC CAC GTC CCG CTG CTT GCC ACC CCC AAC CCT GCC CTC TGAGCGGCCT 2067Asp His Val Pro Leu Leu Ala Thr Pro Asn Pro Ala Leu 510 515 520CTGAGTACCC TGGCGGGAGG CTGGCCCACA CAGAAAGGTG GCAAGAAGAT CGGGAAGACT 2127GAGTAGGGAA GGCAGGGCTG CCCAGAAGTC TCAGAGGCAC CTCACGCCAG CCATCGCGGA 2187GAGCTCAGAG GGCCGTCCCC ACCCTGCCTC CTCCCTGCTG CTTTGCATTC ACTTCCTTGG 2247CCAGAGTCAG GGGACAGGGA GAGAGCTCCA CACTGTAACC ACTGGGTCTG GGCTCCATCC 2307TGCGCCCAAA GACATCCACC CAGACCTCAT TATTTCTTGC TCTATCATTC TGTTTCAATA 2367AAGACATTTG GAATAAACGA GCAAAAAAAA AAAAAAAAAA AAAAAAAAGG GCGGCCGCTC 2427TAGAGGATCC AAGCTTACGT ACGCGTGCAT GCG 2460 520 amino acids amino acidlinear protein internal not provided 2 Met Ala Ser Asp Pro Ile Phe ThrLeu Ala Pro Pro Leu His Cys His 1 5 10 15 Tyr Gly Ala Phe Pro Pro AsnAla Ser Gly Trp Glu Gln Pro Pro Asn 20 25 30 Ala Ser Gly Val Ser Val AlaSer Ala Ala Leu Ala Ala Ser Ala Ala 35 40 45 Ser Arg Val Ala Thr Ser ThrAsp Pro Ser Cys Ser Gly Phe Ala Pro 50 55 60 Pro Asp Phe Asn His Cys LeuLys Asp Trp Asp Tyr Asn Gly Leu Pro 65 70 75 80 Val Leu Thr Thr Asn AlaIle Gly Gln Trp Asp Leu Val Cys Asp Leu 85 90 95 Gly Trp Gln Val Ile LeuGlu Gln Ile Leu Phe Ile Leu Gly Phe Ala 100 105 110 Ser Gly Tyr Leu PheLeu Gly Tyr Pro Ala Asp Arg Phe Gly Arg Arg 115 120 125 Gly Ile Val LeuLeu Thr Leu Gly Leu Val Gly Pro Cys Gly Val Gly 130 135 140 Gly Ala AlaAla Gly Ser Ser Thr Gly Val Met Ala Leu Arg Phe Leu 145 150 155 160 LeuGly Phe Leu Leu Ala Gly Val Asp Leu Gly Val Tyr Leu Met Arg 165 170 175Leu Glu Leu Cys Asp Pro Thr Gln Arg Leu Arg Val Ala Leu Ala Gly 180 185190 Glu Leu Val Gly Val Gly Gly His Phe Leu Phe Leu Gly Leu Ala Leu 195200 205 Val Ser Lys Asp Trp Arg Phe Leu Gln Arg Met Ile Thr Ala Pro Cys210 215 220 Ile Leu Phe Leu Phe Tyr Gly Trp Pro Gly Leu Phe Leu Glu SerAla 225 230 235 240 Arg Trp Leu Ile Val Lys Arg Gln Ile Glu Glu Ala GlnSer Val Leu 245 250 255 Arg Ile Leu Ala Glu Arg Asn Arg Pro His Gly GlnMet Leu Gly Glu 260 265 270 Glu Ala Gln Glu Ala Leu Gln Asp Leu Glu AsnThr Cys Pro Leu Pro 275 280 285 Ala Thr Ser Ser Phe Ser Phe Ala Ser LeuLeu Asn Tyr Arg Asn Ile 290 295 300 Trp Lys Asn Leu Leu Ile Leu Gly PheThr Asn Phe Ile Ala His Ala 305 310 315 320 Ile Arg His Cys Tyr Gln ProVal Gly Gly Gly Gly Ser Pro Ser Asp 325 330 335 Phe Tyr Leu Cys Ser LeuLeu Ala Ser Gly Thr Ala Ala Leu Ala Cys 340 345 350 Val Phe Leu Gly ValThr Val Asp Arg Phe Gly Arg Arg Gly Ile Leu 355 360 365 Leu Leu Ser MetThr Leu Thr Gly Ile Ala Ser Leu Val Leu Leu Gly 370 375 380 Leu Trp AspTyr Leu Asn Glu Ala Ala Ile Thr Thr Phe Ser Val Leu 385 390 395 400 GlyLeu Phe Ser Ser Gln Ala Ala Ala Ile Leu Ser Thr Leu Leu Ala 405 410 415Ala Glu Val Ile Pro Thr Thr Val Arg Gly Arg Gly Leu Gly Leu Ile 420 425430 Met Ala Leu Gly Ala Leu Gly Gly Leu Ser Gly Pro Ala Gln Arg Leu 435440 445 His Met Gly His Gly Ala Phe Leu Gln His Val Val Leu Ala Ala Cys450 455 460 Ala Leu Leu Cys Ile Leu Ser Ile Met Leu Leu Pro Glu Thr LysArg 465 470 475 480 Lys Leu Leu Pro Glu Val Leu Arg Asp Gly Glu Leu CysArg Arg Pro 485 490 495 Ser Leu Leu Arg Gln Pro Pro Pro Thr Arg Cys AspHis Val Pro Leu 500 505 510 Leu Ala Thr Pro Asn Pro Ala Leu 515 520 1490base pairs nucleic acid single linear Genomic DNA not provided CodingSequence 492...1349 3 GTCGACCCAC GCGTCCGGAC CAAGGAGGCG CCCGCGGCTGCAGAGCTGCA GAGCGGGATC 60 TCTTCGAGCT GTCTGTGTCC GGGCAGCCGG CGCGCAACTGAGCCAGAGGA CAGCGCATCC 120 TTTCGGCGCG GGCCGGCAGG GCCCCTGCGG TCGGCAAGCTGGCTCCCCGG GTGGCCACCG 180 GGACCCCCGA GCCCAATGGC GGGGGCGGCG GCAAAATCGACAACACTGTA GAGATCACCC 240 CCACCTCCAA CGGACAGGTC GGGACCCTCG GAGATGCGGTGCCCACGGAG CAGCTGCAGG 300 GTGAGCGGGA GCGCGAGCGG GAGGGGGAGG GAGACGCGGGCGGCGACGGA CTGGGCAGCA 360 GCCTGTCGCT GGCCGTGCCC CCAGGCCCCC TCAGCTTTGAGGCGCTGCTC GCCCAGGTGG 420 GGGCGCTGGG CGGCGGCCAG CAGCTGCAGC TCGGCCTCTGCTGCCTGCCG GTGCTCTTCG 480 TGGCTCTGGG C ATG GCC TCG GAC CCC ATC TTC ACGCTG GCG CCC CCG CTG 530 Met Ala Ser Asp Pro Ile Phe Thr Leu Ala Pro ProLeu 1 5 10 CAT TGC CAC TAC GGG GCC TTC CCC CCT AAT GCC TCT GGC TGG GAGCAG 578 His Cys His Tyr Gly Ala Phe Pro Pro Asn Ala Ser Gly Trp Glu Gln15 20 25 CCT CCC AAT GCC AGC GGC GTC AGC GTC GCC AGC GCT GCC CTA GCA GCC626 Pro Pro Asn Ala Ser Gly Val Ser Val Ala Ser Ala Ala Leu Ala Ala 3035 40 45 AGC GCC GCC AGC CGT GTC GCC ACC AGT ACC GAC CCC TCG TGC AGC GGC674 Ser Ala Ala Ser Arg Val Ala Thr Ser Thr Asp Pro Ser Cys Ser Gly 5055 60 TTC GCC CCG CCG GAC TTC AAC CAT TGC CTC AAG GAT TGG GAC TAT AAT722 Phe Ala Pro Pro Asp Phe Asn His Cys Leu Lys Asp Trp Asp Tyr Asn 6570 75 GGC CTT CCT GTG CTC ACC ACC AAC GCC ATC GGC CAG TGG GAT CTG GTG770 Gly Leu Pro Val Leu Thr Thr Asn Ala Ile Gly Gln Trp Asp Leu Val 8085 90 TGT GAC CTG GGC TGG CAG GTG ATC CTG GAG CAG ATC CTC TTC ATC TTG818 Cys Asp Leu Gly Trp Gln Val Ile Leu Glu Gln Ile Leu Phe Ile Leu 95100 105 GGC TTT GCC TCC GGC TAC CTG TTC CTG GGT TAC CCC GCA GAC AGA TTT866 Gly Phe Ala Ser Gly Tyr Leu Phe Leu Gly Tyr Pro Ala Asp Arg Phe 110115 120 125 GGC CGT CGC GGG ATT GTG CTG CTG ACC TTG GGG CTG GTG GGC CCCTGT 914 Gly Arg Arg Gly Ile Val Leu Leu Thr Leu Gly Leu Val Gly Pro Cys130 135 140 GGA GTA GGA GGG GCT GCT GCA GGC TCC TCC ACA GGC GTC ATG GCCCTC 962 Gly Val Gly Gly Ala Ala Ala Gly Ser Ser Thr Gly Val Met Ala Leu145 150 155 CGA TTC CTC TTG GGC TTT CTG CTT GCC GGT GTT GAC CTG GGT GTCTAC 1010 Arg Phe Leu Leu Gly Phe Leu Leu Ala Gly Val Asp Leu Gly Val Tyr160 165 170 CTG ATG CGC CTG GAG CTG TGC GAC CCA ACC CAG AGG CTT CGG GTGGCC 1058 Leu Met Arg Leu Glu Leu Cys Asp Pro Thr Gln Arg Leu Arg Val Ala175 180 185 CTG GCA GGG GAG TTG GTG GGG GTG GGA GGG CAC TTC CTG TTC CTGGGC 1106 Leu Ala Gly Glu Leu Val Gly Val Gly Gly His Phe Leu Phe Leu Gly190 195 200 205 CTG GCC CTT GTC TCT AAG GAT TGG CGA TTC CTA CAG CGA ATGATC ACC 1154 Leu Ala Leu Val Ser Lys Asp Trp Arg Phe Leu Gln Arg Met IleThr 210 215 220 GCT CCC TGC ATC CTC TTC CTG TTT TAT GGC TGG CCT GGT TTGTTC CTG 1202 Ala Pro Cys Ile Leu Phe Leu Phe Tyr Gly Trp Pro Gly Leu PheLeu 225 230 235 GAG TCC GCA CGG TGG CTG ATA GTG AAG CGG CAG ATT GAG GAGGCT CAG 1250 Glu Ser Ala Arg Trp Leu Ile Val Lys Arg Gln Ile Glu Glu AlaGln 240 245 250 TCT GTG CTG AGG ATC CTG GCT GAG CGA AAC CGG CCC CAT GGGCAG ATG 1298 Ser Val Leu Arg Ile Leu Ala Glu Arg Asn Arg Pro His Gly GlnMet 255 260 265 CTG GGG GAG GAG GCC CAG GAG GCC CTG CAG GAC CTG GAG AGCTCC ACA 1346 Leu Gly Glu Glu Ala Gln Glu Ala Leu Gln Asp Leu Glu Ser SerThr 270 275 280 285 CTG TAACCACTGG GTCTGGGCTC CATCCTGCGC CCAAAGACATCCACCCAGAC 1399 Leu CTCATTATTT CTTGCTCTAT CATTCTGTTT CAATAAAGACATTTGGAATA AACGAGCATA 1459 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA A 1490 286amino acids amino acid linear protein internal not provided 4 Met AlaSer Asp Pro Ile Phe Thr Leu Ala Pro Pro Leu His Cys His 1 5 10 15 TyrGly Ala Phe Pro Pro Asn Ala Ser Gly Trp Glu Gln Pro Pro Asn 20 25 30 AlaSer Gly Val Ser Val Ala Ser Ala Ala Leu Ala Ala Ser Ala Ala 35 40 45 SerArg Val Ala Thr Ser Thr Asp Pro Ser Cys Ser Gly Phe Ala Pro 50 55 60 ProAsp Phe Asn His Cys Leu Lys Asp Trp Asp Tyr Asn Gly Leu Pro 65 70 75 80Val Leu Thr Thr Asn Ala Ile Gly Gln Trp Asp Leu Val Cys Asp Leu 85 90 95Gly Trp Gln Val Ile Leu Glu Gln Ile Leu Phe Ile Leu Gly Phe Ala 100 105110 Ser Gly Tyr Leu Phe Leu Gly Tyr Pro Ala Asp Arg Phe Gly Arg Arg 115120 125 Gly Ile Val Leu Leu Thr Leu Gly Leu Val Gly Pro Cys Gly Val Gly130 135 140 Gly Ala Ala Ala Gly Ser Ser Thr Gly Val Met Ala Leu Arg PheLeu 145 150 155 160 Leu Gly Phe Leu Leu Ala Gly Val Asp Leu Gly Val TyrLeu Met Arg 165 170 175 Leu Glu Leu Cys Asp Pro Thr Gln Arg Leu Arg ValAla Leu Ala Gly 180 185 190 Glu Leu Val Gly Val Gly Gly His Phe Leu PheLeu Gly Leu Ala Leu 195 200 205 Val Ser Lys Asp Trp Arg Phe Leu Gln ArgMet Ile Thr Ala Pro Cys 210 215 220 Ile Leu Phe Leu Phe Tyr Gly Trp ProGly Leu Phe Leu Glu Ser Ala 225 230 235 240 Arg Trp Leu Ile Val Lys ArgGln Ile Glu Glu Ala Gln Ser Val Leu 245 250 255 Arg Ile Leu Ala Glu ArgAsn Arg Pro His Gly Gln Met Leu Gly Glu 260 265 270 Glu Ala Gln Glu AlaLeu Gln Asp Leu Glu Ser Ser Thr Leu 275 280 285 1411 base pairs nucleicacid single linear cDNA not provided Coding Sequence 1...966 5 GTC GACCCA CGC GTC CGG GGC CTG GCC CTT GTC TCT AAG GAC TGG CGG 48 Val Asp ProArg Val Arg Gly Leu Ala Leu Val Ser Lys Asp Trp Arg 1 5 10 15 TTC CTGCAG CGA ATG ATC ACC GCT CCT TGC ATC CTC TTC CTG TTT TAT 96 Phe Leu GlnArg Met Ile Thr Ala Pro Cys Ile Leu Phe Leu Phe Tyr 20 25 30 GGC TGG CCCGGT CTG TTT CTG GAC TCC GCA CGG TGG CTG ATA GTG AAA 144 Gly Trp Pro GlyLeu Phe Leu Asp Ser Ala Arg Trp Leu Ile Val Lys 35 40 45 CGG CAG ATT GAGGAA GCC CAG TCT GTG CTG AGG ATC CTG GCT GAG CGA 192 Arg Gln Ile Glu GluAla Gln Ser Val Leu Arg Ile Leu Ala Glu Arg 50 55 60 AAC CGG CCC CAT GGCCAG ATG CTG GGA GAA GAG GCC CAG GAA GCC CTG 240 Asn Arg Pro His Gly GlnMet Leu Gly Glu Glu Ala Gln Glu Ala Leu 65 70 75 80 CAG GAG CTG GAG AATACC TGT CCT CTC CCC ACA ACG TCC ACC TTT TCC 288 Gln Glu Leu Glu Asn ThrCys Pro Leu Pro Thr Thr Ser Thr Phe Ser 85 90 95 TTC GCC TCC CTC CTC AACTAC CGA AAC ATC TGG AAA AAT CTG CTT ATC 336 Phe Ala Ser Leu Leu Asn TyrArg Asn Ile Trp Lys Asn Leu Leu Ile 100 105 110 CTG GGC TTC ACC AAC TTTATC GCC CAT GCC ATT CGC CAC TGC TAC CAG 384 Leu Gly Phe Thr Asn Phe IleAla His Ala Ile Arg His Cys Tyr Gln 115 120 125 CCT GTG GGA GGA GGA GGGAGC CCA TCA GAC TTC TAC TTG TGC TCT CTT 432 Pro Val Gly Gly Gly Gly SerPro Ser Asp Phe Tyr Leu Cys Ser Leu 130 135 140 CTG GCC AGC GGC ACA GCAGCC CTG GCC TGC GTC TTC CTG GGG GTG ACC 480 Leu Ala Ser Gly Thr Ala AlaLeu Ala Cys Val Phe Leu Gly Val Thr 145 150 155 160 GTG GAC CGT TTC GGCCGT CGG GGC ATC CTG CTT CTC TCA ATG ACT CTC 528 Val Asp Arg Phe Gly ArgArg Gly Ile Leu Leu Leu Ser Met Thr Leu 165 170 175 ACG GGG ATT GCA TCCCTG GTC TTG CTG GGC CTG TGG GAT TAT CTG AAC 576 Thr Gly Ile Ala Ser LeuVal Leu Leu Gly Leu Trp Asp Tyr Leu Asn 180 185 190 GAT GCT GCC ATC ACAACC TTC TCG GTC CTC GGA CTC TTC TCC TCC CAA 624 Asp Ala Ala Ile Thr ThrPhe Ser Val Leu Gly Leu Phe Ser Ser Gln 195 200 205 GCT TCT GCT ATC CTCAGT ACC CTC CTT GCT GCT GAA GTC ATC CCC ACC 672 Ala Ser Ala Ile Leu SerThr Leu Leu Ala Ala Glu Val Ile Pro Thr 210 215 220 ACT GTC CGG GGC CGTGGC CTG GGC CTT ATC ATG GCA CTT GGG GCG CTT 720 Thr Val Arg Gly Arg GlyLeu Gly Leu Ile Met Ala Leu Gly Ala Leu 225 230 235 240 GGA GGG CTG AGCTGT CCA GCT CAG CGC CTC CAC ATG GGC CAT GGA GCT 768 Gly Gly Leu Ser CysPro Ala Gln Arg Leu His Met Gly His Gly Ala 245 250 255 TTC CTG CAG CATGTG GTA CTG GCG GCC TGT GCC CTC CTC TGC ATC CTT 816 Phe Leu Gln His ValVal Leu Ala Ala Cys Ala Leu Leu Cys Ile Leu 260 265 270 AGC ATC ATG CTGCTG CCA GAG ACC AAG CGC AAG CTT CTG CCA GAG GTA 864 Ser Ile Met Leu LeuPro Glu Thr Lys Arg Lys Leu Leu Pro Glu Val 275 280 285 CTC CGG GAT GGGGAA CTG TGC CGT CGG CCT TCC CTG CTG AGG CAG CCA 912 Leu Arg Asp Gly GluLeu Cys Arg Arg Pro Ser Leu Leu Arg Gln Pro 290 295 300 CCT CCT AAC CGCTGT GAC CAT GTC CCC CTG CTA GCC ACT CCT AAT CCT 960 Pro Pro Asn Arg CysAsp His Val Pro Leu Leu Ala Thr Pro Asn Pro 305 310 315 320 GCC CTCTAAGCAGCCT CTGAGCCTGG TGGGAGGCTG GCCATTTAGA AAGGTGACGG 1016 Ala LeuAGGGCTGGCT AGCAAGATAG ACGGAGAGGC AAGGCCACCC TGTACATACA AAAGGCTCCA 1076AGGCGCCTCA CGCCATCTAG GAGAGCTCAG AGTGCCATTC CCAACCCCCT CTCCTCCCCG 1136CTGCTTTCTG TTCACTTCAT CAGCAAGAGT CAGGACGGGG ATAGCATCTC GCTATAACCG 1196TTAGGTCTGG GATCCATCCT ATGCCCAAAG ACATTTTCCC AGACCTTGCT CTTTCTCGCT 1256CTTCATTCTG TTTCAATAAA AGACATTTTG AATAAATGAG CATTTCATAG CCTGGGAAAA 1316AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1376AAAAAAAAAA AAAAAAAAAA AAAAAGGGCG GCCGC 1411 322 amino acids amino acidlinear protein internal not provided 6 Val Asp Pro Arg Val Arg Gly LeuAla Leu Val Ser Lys Asp Trp Arg 1 5 10 15 Phe Leu Gln Arg Met Ile ThrAla Pro Cys Ile Leu Phe Leu Phe Tyr 20 25 30 Gly Trp Pro Gly Leu Phe LeuAsp Ser Ala Arg Trp Leu Ile Val Lys 35 40 45 Arg Gln Ile Glu Glu Ala GlnSer Val Leu Arg Ile Leu Ala Glu Arg 50 55 60 Asn Arg Pro His Gly Gln MetLeu Gly Glu Glu Ala Gln Glu Ala Leu 65 70 75 80 Gln Glu Leu Glu Asn ThrCys Pro Leu Pro Thr Thr Ser Thr Phe Ser 85 90 95 Phe Ala Ser Leu Leu AsnTyr Arg Asn Ile Trp Lys Asn Leu Leu Ile 100 105 110 Leu Gly Phe Thr AsnPhe Ile Ala His Ala Ile Arg His Cys Tyr Gln 115 120 125 Pro Val Gly GlyGly Gly Ser Pro Ser Asp Phe Tyr Leu Cys Ser Leu 130 135 140 Leu Ala SerGly Thr Ala Ala Leu Ala Cys Val Phe Leu Gly Val Thr 145 150 155 160 ValAsp Arg Phe Gly Arg Arg Gly Ile Leu Leu Leu Ser Met Thr Leu 165 170 175Thr Gly Ile Ala Ser Leu Val Leu Leu Gly Leu Trp Asp Tyr Leu Asn 180 185190 Asp Ala Ala Ile Thr Thr Phe Ser Val Leu Gly Leu Phe Ser Ser Gln 195200 205 Ala Ser Ala Ile Leu Ser Thr Leu Leu Ala Ala Glu Val Ile Pro Thr210 215 220 Thr Val Arg Gly Arg Gly Leu Gly Leu Ile Met Ala Leu Gly AlaLeu 225 230 235 240 Gly Gly Leu Ser Cys Pro Ala Gln Arg Leu His Met GlyHis Gly Ala 245 250 255 Phe Leu Gln His Val Val Leu Ala Ala Cys Ala LeuLeu Cys Ile Leu 260 265 270 Ser Ile Met Leu Leu Pro Glu Thr Lys Arg LysLeu Leu Pro Glu Val 275 280 285 Leu Arg Asp Gly Glu Leu Cys Arg Arg ProSer Leu Leu Arg Gln Pro 290 295 300 Pro Pro Asn Arg Cys Asp His Val ProLeu Leu Ala Thr Pro Asn Pro 305 310 315 320 Ala Leu 10 amino acids aminoacid linear peptide not provided 7 Gly Tyr Xaa Xaa Asp Arg Xaa Gly ArgArg 1 5 10 10 amino acids amino acid linear peptide not provided 8 GlyTyr Pro Ala Asp Arg Phe Gly Arg Arg 1 5 10 5 amino acids amino acidlinear peptide not provided 9 Ile Ser Lys Met Phe 1 5 28 amino acidsamino acid linear peptide not provided 10 Arg Xaa Leu Xaa Gly Xaa XaaLeu Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Leu Xaa Thr Glu TrpXaa Xaa Xaa Xaa Xaa Arg 20 25 28 amino acids amino acid linear peptidenot provided 11 Arg Phe Leu Leu Gly Phe Leu Leu Ala Gly Val Asp Leu GlyVal Tyr 1 5 10 15 Leu Met Arg Leu Glu Leu Cys Asp Pro Thr Gln Arg 20 257 amino acids amino acid linear peptide not provided 12 Pro Glu Ser XaaArg Trp Leu 1 5 7 amino acids amino acid linear peptide not provided 13Leu Glu Ser Ala Arg Trp Leu 1 5 6 amino acids amino acid linear peptidenot provided 14 Leu Leu Pro Glu Thr Lys 1 5 4 amino acids amino acidlinear peptide not provided 15 Thr Gln Thr Arg 1 15 amino acids aminoacid linear peptide not provided 16 Leu Xaa Asn Xaa Glu Leu Tyr Pro ThrXaa Xaa Arg Asn Leu Gly 1 5 10 15 15 amino acids amino acid linearpeptide not provided 17 Leu Leu Ala Ala Glu Val Ile Pro Thr Thr Val ArgGly Arg Gly 1 5 10 15

What is claimed is:
 1. An isolated polypeptide comprising the amino acidsequence of SEQ ID NO:2.
 2. An isolated polypeptide comprising the aminoacid sequence of SEQ ID NO:4.
 3. An isolated polypeptide comprising theamino acid sequence of SEQ ID NO:6.
 4. An isolated polypeptideconsisting of the amino acid sequence of SEQ ID NO:2.
 5. An isolatedpolypeptide consisting of the amino acid sequence of SEQ ID NO:4.
 6. Anisolated polypeptide consisting of the amino acid sequence of SEQ IDNO:6.
 7. The isolated pure polypeptide of claim 1 wherein thepolypeptide is a fusion polypeptide.
 8. The isolated pure polypeptide ofclaim 2 wherein the polypeptide is a fusion polypeptide.
 9. The isolatedpure polypeptide of claim 3 wherein the polypeptide is a fusionpolypeptide.